Patent Publication Number: US-2023151433-A1

Title: Detecting melanoma

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     The present application claims priority to U.S. Provisional Patent Application No. 63/019,753, filed May 4, 2020, which is hereby incorporated by reference in its entirety. 
    
    
     FIELD OF INVENTION 
     Provided herein is technology for primary cutaneous melanoma screening and particularly, but not exclusively, to methods, compositions, and related uses for detecting the presence of primary cutaneous melanoma. 
     BACKGROUND 
     Melanoma is a serious form of skin cancer in humans. It arises from the pigment cells (melanocytes), usually in the skin. The incidence of melanoma is increasing at the fastest rate of all cancers in the United States with a lifetime risk of 1 in 68. Although melanoma accounts for only 4% of all dermatologic cancers, it is responsible for 80% of all deaths from skin cancers. It has long been realized that recognition and diagnosis of melanoma, when it is early stage disease, is key to its cure. There are several types of melanoma, defined by where they first appear, including skin and eye melanoma and in rare instances in the GI tract or lymph nodes. 
     Primary cutaneous melanoma (PCM) is a common life-threatening malignancy responsible for 80% of skin cancer-related deaths (see, Miller, A. J. and M. C. Mihm Jr, New England Journal of Medicine, 2006. 355(1): p. 51-65). While the number of PCM diagnoses is ever increasing with a projected 150,000+ cases (in situ and invasive) in 2017 (American Cancer Society, 2017), the prognosis depends upon disease stage at diagnosis with a five-year overall survival rate of 98.1% for those with localized disease, 63.6% for those with regional disease, and 16.1% for those with distant metastases (American Cancer Society, 2017). Current disease surveillance of pre-metastatic PCM patients requires in-office visits with health care providers and interval PET/CT imaging. The cost of care already exceeds the resources of many patients with payers unable or unwilling to reimburse PET/CT. Lower socioeconomic status (SES) as evidenced by Medicaid insurance has been shown to delay melanoma care substantially (see, Adamson, A. S., et al., JAMA Dermatol, 2017. 153(11): p. 1106-1113). 
     As sophisticated hospital-based imaging procedures will only increase in price over time there is a need for novel affordable outpatient surveillance solutions such as those described in this application. Indeed, to lessen the heavy toll of melanoma, effective screening approaches are urgently needed. There is an imperative for innovation that will deliver accurate, affordable, and safe screening tools for the pre-symptomatic detection of earliest stage of melanoma and advanced melanoma. 
     The present invention addresses such needs. Indeed, the present invention provides novel methylated DNA markers that discriminate cases of melanoma and its various subtypes (e.g., metastatic melanoma, primary melanoma) within various biological samples (e.g., tissue, blood). 
     SUMMARY 
     Blood tests for melanoma screening have typically been based on marker detection in the clear portion of blood (plasma or serum) and have been insensitive or nonspecific for earliest stage disease. To address these historical deficiencies, experiments conducted herein explored a rational new approach anchored on discriminant methylated DNA markers. Marker discovery and validation occurred through utilization of high quality flash-frozen bio-banked tissues. Feasibility of marker detection in blood compartments was established by use of an exquisitely sensitive analytical platform (e.g., a quantitative methylation-specific PCR; qMSP). Such results indicate that this blood testing approach establishes a requisite analytical sensitivity to detect curable-stage cancers while accurately predicting tumor site and avoiding frequent false positives. 
     Methylated DNA has been studied as a potential class of biomarkers in the tissues of most tumor types. In many instances, DNA methyltransferases add a methyl group to DNA at cytosine-phosphate-guanine (CpG) island sites as an epigenetic control of gene expression. In a biologically attractive mechanism, acquired methylation events in promoter regions of tumor suppressor genes are thought to silence expression, thus contributing to oncogenesis. DNA methylation may be a more chemically and biologically stable diagnostic tool than RNA or protein expression (Laird (2010) Nat Rev Genet 11: 191-203). Furthermore, in other cancers like sporadic colon cancer, methylation markers offer excellent specificity and are more broadly informative and sensitive than individual DNA mutations (Zou et al (2007) Cancer Epidemiol Biomarkers Prev 16: 2686-96). 
     Analysis of CpG islands has yielded important findings when applied to animal models and human cell lines. For example, Zhang and colleagues found that amplicons from different parts of the same CpG island may have different levels of methylation (Zhang et al. (2009) PLoS Genet 5: e1000438). Further, methylation levels were distributed bi-modally between highly methylated and unmethylated sequences, further supporting the binary switch-like pattern of DNA methyltransferase activity (Zhang et al. (2009) PLoS Genet 5: e1000438). Analysis of murine tissues in vivo and cell lines in vitro demonstrated that only about 0.3% of high CpG density promoters (HCP, defined as having &gt;7% CpG sequence within a 300 base pair region) were methylated, whereas areas of low CpG density (LCP, defined as having &lt;5% CpG sequence within a 300 base pair region) tended to be frequently methylated in a dynamic tissue-specific pattern (Meissner et al. (2008) Nature 454: 766-70). HCPs include promoters for ubiquitous housekeeping genes and highly regulated developmental genes. Among the HCP sites methylated at &gt;50% were several established markers such as Wnt 2, NDRG2, SFRP2, and BMP3 (Meissner et al. (2008) Nature 454: 766-70). 
     Epigenetic methylation of DNA at cytosine-phosphate-guanine (CpG) island sites by DNA methyltransferases has been studied as a potential class of biomarkers in the tissues of most tumor types. 
     Several methods are available to search for novel methylation markers. While micro-array-based interrogation of CpG methylation is a reasonable, high-throughput approach, this strategy is biased towards known regions of interest, mainly established tumor suppressor promotors. Alternative methods for genome-wide analysis of DNA methylation have been developed in the last decade. There are four basic approaches. The first employs digestion of DNA by restriction enzymes which recognize specific methylated sites, followed by several possible analytic techniques which provide methylation data limited to the enzyme recognition site or the primers used to amplify the DNA in quantification steps (such as methylation-specific PCR; MSP). A second approach enriches methylated fractions of genomic DNA using anti-bodies directed to methyl-cytosine or other methylation-specific binding domains followed by microarray analysis or sequencing to map the fragment to a reference genome. This approach does not provide single nucleotide resolution of all methylated sites within the fragment. A third approach begins with bisulfite treatment of the DNA to convert all unmethylated cytosines to uracil, followed by restriction enzyme digestion and complete sequencing of all fragments after coupling to an adapter ligand. The choice of restriction enzymes can enrich the fragments for CpG dense regions, reducing the number of redundant sequences which may map to multiple gene positions during analysis. A fourth approach involves a bisulfite-free treatment of the DNA that describe a bisulfite-free and base-resolution sequencing method, TET-assisted pyridine borane sequencing (TAPS), for non-destructive and direct detection of 5-methylcytosine and 5-hydroxymethylcytosine without affecting unmodified cytosines (see, Liu et al., 2019, Nat Biotechnol. 37, pp. 424-429). In some embodiments, regardless of the specific enzymatic conversion approach, only the methylated cytosines are converted. 
     Reduced Representation Bisulfite Sequencing (RRBS) yields CpG methylation status data at single nucleotide resolution of 80-90% of all CpG islands and a majority of tumor suppressor promoters at medium to high read coverage. In cancer case—control studies, analysis of these reads results in the identification of differentially methylated regions (DMRs). In previous RRBS analysis of pancreatic cancer specimens, hundreds of DMRs were uncovered, many of which had never been associated with carcinogenesis and many of which were unannotated. Further validation studies on independent tissue sample sets confirmed marker CpGs which were 100% sensitive and specific in terms of performance. 
     Provided herein is technology for melanoma and various melanoma subtypes (e.g., metastatic melanoma, primary cutaneous melanoma) screening and particularly, but not exclusively, to methods, compositions, and related uses for detecting the presence of melanoma and various melanoma subtypes (e.g., metastatic melanoma, primary cutaneous melanoma). 
     Indeed, as described in Examples I and II, experiments conducted during the course for identifying embodiments for the present invention identified a novel set of differentially methylated regions (DMRs) for discriminating 1) melanoma derived DNA from non-neoplastic control tissue, 2) DNA derived from metastatic melanoma tissue from non-neoplastic control DNA, and 3) DNA derived from primary cutaneous melanoma tissue from non-neoplastic control DNA. 
     Such experiments list and describe 331 novel DNA methylation markers distinguishing melanoma tissue from benign tissue (see, Tables 1A, 1B, 4, 5A, 5B, 5C, 7A, and 7B; Examples I and II), metastatic melanoma tissue from benign tissue (see, Tables 5C; Example I), primary cutaneous melanoma tissue from benign tissue (see, Tables 5C; Example I), and detecting melanoma within a blood sample (see, Tables 2A, 2B, 4, 5A, 6 and 8A; Examples I and II). 
     From these 331 novel DNA methylation markers, further experiments identified the following markers and/or panels of markers capable of distinguishing melanoma tissue from benign tissue:
         c10orf55, c2orf82, FOXL2NB, CLIC5, FAM174B_B, FLJ22536_A, FOXP1, GALNT3_A, HOPX, HOXA9_A, ITPKA, KCNQ4_A, LMX1B, MAX.chr1.110627096-110627364, MAX.chr10.22541869-22541953, MAX.chr10.62492690-62492812, MAX.chr13.29106812-29106917, MAX.chr17.73073682-73073830, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, MAX.chr5.42992655-42992768, MAX.chr5.60921627-60921853, MAX.chr6.29521537-29521696, MAX.chr7.155259597-155259763, ME3, MIR155HG, NFATC2_A, OCA2, OXT, RNF207_A, RUNX2, SIX4_A, STX16, TAL1, TMEM30B, and TSPAN33 (see, Table 4, 5A, 5B; Example I);   AGRN, BMP8B, c17orf46_B, c17orf57_B, C2CD4A, C2CD4D_B, CDYL, CMAH, DENND2D, DLEU2, DSCR6, FLJ22536_B, FLJ45983, FOXF2, GALNT3_B, HCG4P6, HOXA9_B, KIFC2, LDLRAD2, LY75, LYL1, LYN, MAPK13, MAX.chr1.1072486-1072508, MAX.chr1.32237693-32237785, MAX.chr10.62492374-62492793, MAX.chr11.14926535-14926715, MAX.chr16.54970444-54970469, MAX.chr19.16439332-16439390, MAX.chr20.21080670-21081280, MAX.chr4.113432264-113432298, MAX.chr7.129425668-129425719, MAX.chr8.82543163-82543213, PARP15, PRKAG2, PROC_A, PROM1_B, PTGER4_A, PTP4A3, SDCCAG8, SH3PXD2A, SLC2A2, SLC35D3, TBR1, TBX2, TCP11, TFAP2A, and TRIM73 (see, Table 7A and 7B; Example II);   MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-73073830, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22541874-22541948, ME3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763 (see, Tables 9, 10, 11; Example II).       

     From these 331 novel DNA methylation markers, further experiments identified the following markers and/or panels of markers capable of distinguishing metastatic melanoma tissue from benign tissue:
         MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763, FOXL2NB, and HOXA9_A (see, Table 5C, Example I); and   MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-73073830, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22541874-22541948, ME3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763 (see, Tables 9, 10, 11; Example II).       

     From these 331 novel DNA methylation markers, further experiments identified the following markers and/or panels of markers capable of distinguishing primary cutaneous melanoma tissue from benign tissue:
         MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763, FOXL2NB, and HOXA9_A (see, Table 5C, Example I);   MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-73073830, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22541874-22541948, ME3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763 (see, Tables 9, 10, 11; Example II).       

     From these 331 novel DNA methylation markers, further experiments identified the following markers and/or panels of markers for detecting melanoma in blood samples (e.g., plasma samples, whole blood samples, leukocyte samples, serum samples):
         c10orf55, c2orf82, FOXL2NB, CLIC5, FAM174B_B, FLJ22536_A, FOXP1, GALNT3_A, HOPX, HOXA9_A, ITPKA, KCNQ4_A, LMX1B, MAX.chr1.110627096-110627364, MAX.chr10.22541869-22541953, MAX.chr10.62492690-62492812, MAX.chr13.29106812-29106917, MAX.chr17.73073682-73073830, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, MAX.chr5.42992655-42992768, MAX.chr5.60921627-60921853, MAX.chr6.29521537-29521696, MAX.chr7.155259597-155259763, ME3, MIR155HG, NFATC2_A, OCA2, OXT, RNF207_A, RUNX2, SIX4_A, STX16, TAL1, TMEM30B, and TSPAN33 (see, Table 4, 5A, 5B, 6; Example I);   one or more of the markers recited in Tables 8A and 8B; and   MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-73073830, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22541874-22541948, ME3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763 (see, Tables 9, 10, 11; Example II).       

     As described herein, the technology provides a number of methylated DNA markers and subsets thereof (e.g., sets of 2, 3, 4, 5, 6, 7, or 8 markers) with high discrimination for melanoma overall and various types of melanoma (e.g., metastatic melanoma, primary cutaneous melanoma). Experiments applied a selection filter to candidate markers to identify markers that provide a high signal to noise ratio and a low background level to provide high specificity for purposes of melanoma screening or diagnosis. 
     In some embodiments, the technology is related to assessing the presence of and methylation state of one or more of the markers identified herein in a biological sample (e.g., melanoma tissue, plasma sample). These markers comprise one or more differentially methylated regions (DMR) as discussed herein, e.g., as provided in Tables 1A, 2A, 7A, 8A, and 9. Methylation state is assessed in embodiments of the technology. As such, the technology provided herein is not restricted in the method by which a gene&#39;s methylation state is measured. For example, in some embodiments the methylation state is measured by a genome scanning method. For example, one method involves restriction landmark genomic scanning (Kawai et al. (1994)  Mol. Cell. Biol.  14: 7421-7427) and another example involves methylation-sensitive arbitrarily primed PCR (Gonzalgo et al. (1997)  Cancer Res.  57: 594-599). In some embodiments, changes in methylation patterns at specific CpG sites are monitored by digestion of genomic DNA with methylation-sensitive restriction enzymes followed by Southern analysis of the regions of interest (digestion-Southern method). In some embodiments, analyzing changes in methylation patterns involves a PCR-based process that involves digestion of genomic DNA with methylation-sensitive restriction enzymes or methylation-dependent restriction enzymes prior to PCR amplification (Singer-Sam et al. (1990)  Nucl. Acids Res.  18: 687). In addition, other techniques have been reported that utilize bisulfite treatment of DNA as a starting point for methylation analysis. These include methylation-specific PCR (MSP) (Herman et al. (1992)  Proc. Natl. Acad. Sci. USA  93: 9821-9826) and restriction enzyme digestion of PCR products amplified from bisulfite-converted DNA (Sadri and Hornsby (1996)  Nucl. Acids Res.  24: 5058-5059; and Xiong and Laird (1997)  Nucl. Acids Res.  25: 2532-2534). PCR techniques have been developed for detection of gene mutations (Kuppuswamy et al. (1991)  Proc. Natl. Acad. Sci. USA  88: 1143-1147) and quantification of allelic-specific expression (Szabo and Mann (1995)  Genes Dev.  9: 3097-3108; and Singer-Sam et al. (1992)  PCR Methods Appl.  1: 160-163). Such techniques use internal primers, which anneal to a PCR-generated template and terminate immediately 5′ of the single nucleotide to be assayed. Methods using a “quantitative Ms-SNuPE assay” as described in U.S. Pat. No. 7,037,650 are used in some embodiments. 
     Upon evaluating a methylation state, the methylation state is often expressed as the fraction or percentage of individual strands of DNA that is methylated at a particular site (e.g., at a single nucleotide, at a particular region or locus, at a longer sequence of interest, e.g., up to a ˜100-bp, 200-bp, 500-bp, 1000-bp subsequence of a DNA or longer) relative to the total population of DNA in the sample comprising that particular site. Traditionally, the amount of the unmethylated nucleic acid is determined by PCR using calibrators. Then, a known amount of DNA is bisulfite treated (or non-bisulfite treated (see, Liu et al., 2019, Nat Biotechnol. 37, pp. 424-429)) and the resulting methylation-specific sequence is determined using either a real-time PCR or other exponential amplification, e.g., a QuARTS assay (e.g., as provided by U.S. Pat. Nos. 8,361,720; 8,715,937; 8,916,344; and 9,212,392). 
     For example, in some embodiments. methods comprise generating a standard curve for the unmethylated target by using external standards. The standard curve is constructed from at least two points and relates the real-time Ct value for unmethylated DNA to known quantitative standards. Then, a second standard curve for the methylated target is constructed from at least two points and external standards. This second standard curve relates the Ct for methylated DNA to known quantitative standards. Next, the test sample Ct values are determined for the methylated and unmethylated populations and the genomic equivalents of DNA are calculated from the standard curves produced by the first two steps. The percentage of methylation at the site of interest is calculated from the amount of methylated DNAs relative to the total amount of DNAs in the population, e.g., (number of methylated DNAs)/(the number of methylated DNAs+number of unmethylated DNAs)×100. 
     In some embodiments, the plurality of different target regions comprise a reference target region, and in certain preferred embodiments, the reference target region comprises 3-actin and/or ZDHHC1, and/or B3GALT6. 
     Also provided herein are compositions and kits for practicing the methods. For example, in some embodiments, reagents (e.g., primers, probes) specific for one or more MDMs are provided alone or in sets (e.g., sets of primers pairs for amplifying a plurality of markers). Additional reagents for conducting a detection assay may also be provided (e.g., enzymes, buffers, positive and negative controls for conducting QuARTS, PCR, sequencing, bisulfite, Ten-Eleven Translocation (TET) enzyme (e.g., human TET1, human TET2, human TET3, murine TET1, murine TET2, murine TET3,  Naegleria  TET (NgTET),  Coprinopsis cinerea  (CcTET)), or a variant thereof), organic borane, or other assays). In some embodiments, the kits contain a reagent capable of modifying DNA in a methylation-specific manner (e.g., a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, Ten-Eleven Translocation (TET) enzyme (e.g., human TET1, human TET2, human TET3, murine TET1, murine TET2, murine TET3,  Naegleria  TET (NgTET),  Coprinopsis cinerea  (CcTET)), or a variant thereof), organic borane), and/or an agent capable of detecting an increased level of a protein marker described herein. In some embodiments, the kits containing one or more reagents necessary, sufficient, or useful for conducting a method are provided. Also provided are reaction mixtures containing the reagents. Further provided are master mix reagent sets containing a plurality of reagents that may be added to each other and/or to a test sample to complete a reaction mixture. 
     In some embodiments, the technology described herein is associated with a programmable machine designed to perform a sequence of arithmetic or logical operations as provided by the methods described herein. For example, some embodiments of the technology are associated with (e.g., implemented in) computer software and/or computer hardware. In one aspect, the technology relates to a computer comprising a form of memory, an element for performing arithmetic and logical operations, and a processing element (e.g., a microprocessor) for executing a series of instructions (e.g., a method as provided herein) to read, manipulate, and store data. In some embodiments, a microprocessor is part of a system for determining a methylation state (e.g., of one or more DMR, e.g., DMR 1-331 as provided in Tables 1A, 2A, 7A, 8A, and 9); comparing methylation states (e.g., of one or more DMR, e.g., DMR 1-331 as provided in Tables 1A, 2A, 7A, 8A, and 9); generating standard curves; determining a Ct value; calculating a fraction, frequency, or percentage of methylation (e.g., of one or more DMR, e.g., DMR 1-331 as provided in Tables 1A, 2A, 7A, 8A, and 9); identifying a CpG island; determining a specificity and/or sensitivity of an assay or marker; calculating an ROC curve and an associated AUC; sequence analysis; all as described herein or is known in the art. 
     In some embodiments, a microprocessor or computer uses methylation state data in an algorithm to predict a site of a cancer. 
     In some embodiments, a software or hardware component receives the results of multiple assays and determines a single value result to report to a user that indicates a cancer risk based on the results of the multiple assays (e.g., determining the methylation state of multiple DMR, e.g., as provided in Tables 1A, 2A, 7A, 8A, and 9). Related embodiments calculate a risk factor based on a mathematical combination (e.g., a weighted combination, a linear combination) of the results from multiple assays, e.g., determining the methylation states of multiple markers (such as multiple DMR, e.g., as provided in Tables 1A, 2A, 7A, 8A, and 9). In some embodiments, the methylation state of a DMR defines a dimension and may have values in a multidimensional space and the coordinate defined by the methylation states of multiple DMR is a result, e.g., to report to a user, e.g., related to a cancer risk. 
     Some embodiments comprise a storage medium and memory components. Memory components (e.g., volatile and/or nonvolatile memory) find use in storing instructions (e.g., an embodiment of a process as provided herein) and/or data (e.g., a work piece such as methylation measurements, sequences, and statistical descriptions associated therewith). Some embodiments relate to systems also comprising one or more of a CPU, a graphics card, and a user interface (e.g., comprising an output device such as display and an input device such as a keyboard). 
     Programmable machines associated with the technology comprise conventional extant technologies and technologies in development or yet to be developed (e.g., a quantum computer, a chemical computer, a DNA computer, an optical computer, a spintronics based computer, etc.). 
     In some embodiments, the technology comprises a wired (e.g., metallic cable, fiber optic) or wireless transmission medium for transmitting data. For example, some embodiments relate to data transmission over a network (e.g., a local area network (LAN), a wide area network (WAN), an ad-hoc network, the internet, etc.). In some embodiments, programmable machines are present on such a network as peers and in some embodiments the programmable machines have a client/server relationship. 
     In some embodiments, data are stored on a computer-readable storage medium such as a hard disk, flash memory, optical media, a floppy disk, etc. 
     In some embodiments, the technology provided herein is associated with a plurality of programmable devices that operate in concert to perform a method as described herein. For example, in some embodiments, a plurality of computers (e.g., connected by a network) may work in parallel to collect and process data, e.g., in an implementation of cluster computing or grid computing or some other distributed computer architecture that relies on complete computers (with onboard CPUs, storage, power supplies, network interfaces, etc.) connected to a network (private, public, or the internet) by a conventional network interface, such as Ethernet, fiber optic, or by a wireless network technology. 
     For example, some embodiments provide a computer that includes a computer-readable medium. The embodiment includes a random access memory (RAM) coupled to a processor. The processor executes computer-executable program instructions stored in memory. Such processors may include a microprocessor, an ASIC, a state machine, or other processor, and can be any of a number of computer processors, such as processors from Intel Corporation of Santa Clara, Calif. and Motorola Corporation of Schaumburg, Ill. Such processors include, or may be in communication with, media, for example computer-readable media, which stores instructions that, when executed by the processor, cause the processor to perform the steps described herein. 
     Embodiments of computer-readable media include, but are not limited to, an electronic, optical, magnetic, or other storage or transmission device capable of providing a processor with computer-readable instructions. Other examples of suitable media include, but are not limited to, a floppy disk, CD-ROM, DVD, magnetic disk, memory chip, ROM, RAM, an ASIC, a configured processor, all optical media, all magnetic tape or other magnetic media, or any other medium from which a computer processor can read instructions. Also, various other forms of computer-readable media may transmit or carry instructions to a computer, including a router, private or public network, or other transmission device or channel, both wired and wireless. The instructions may comprise code from any suitable computer-programming language, including, for example, C, C++, C#, Visual Basic, Java, Python, Perl, and JavaScript. 
     Computers are connected in some embodiments to a network. Computers may also include a number of external or internal devices such as a mouse, a CD-ROM, DVD, a keyboard, a display, or other input or output devices. Examples of computers are personal computers, digital assistants, personal digital assistants, cellular phones, mobile phones, smart phones, pagers, digital tablets, laptop computers, internet appliances, and other processor-based devices. In general, the computers related to aspects of the technology provided herein may be any type of processor-based platform that operates on any operating system, such as Microsoft Windows, Linux, UNIX, Mac OS X, etc., capable of supporting one or more programs comprising the technology provided herein. Some embodiments comprise a personal computer executing other application programs (e.g., applications). The applications can be contained in memory and can include, for example, a word processing application, a spreadsheet application, an email application, an instant messenger application, a presentation application, an Internet browser application, a calendar/organizer application, and any other application capable of being executed by a client device. 
     All such components, computers, and systems described herein as associated with the technology may be logical or virtual. 
     Accordingly, provided herein is technology related to a method of screening for melanoma and/or various forms of melanoma (e.g., metastatic melanoma, primary cutaneous melanoma) in a sample obtained from a subject, the method comprising assaying a methylation state of a marker in a sample obtained from a subject (e.g., melanoma tissue) (e.g., tissue sample, plasma sample) and identifying the subject as having melanoma and/or various forms of melanoma (e.g., metastatic melanoma, primary cutaneous melanoma) when the methylation state of the marker is different than a methylation state of the marker assayed in a subject that does not have such cancer, wherein the marker comprises a base in a differentially methylated region (DMR) selected from a group consisting of DMR 1-331 as provided in Tables 1A, 2A, 7A, 8A, and 9. 
     In some embodiments wherein the sample obtained from the subject is skin tissue and the methylation state of one or more of the following markers is different than a methylation state of the one or more markers assayed in a subject that does not have melanoma indicates the subject has melanoma: c10orf55, c2orf82, FOXL2NB, CLIC5, FAM174B_B, FLJ22536_A, FOXP1, GALNT3_A, HOPX, HOXA9_A, ITPKA, KCNQ4_A, LMX1B, MAX.chr1.110627096-110627364, MAX.chr10.22541869-22541953, MAX.chr10.62492690-62492812, MAX.chr13.29106812-29106917, MAX.chr17.73073682-73073830, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, MAX.chr5.42992655-42992768, MAX.chr5.60921627-60921853, MAX.chr6.29521537-29521696, MAX.chr7.155259597-155259763, ME3, MIR155HG, NFATC2_A, OCA2, OXT, RNF207_A, RUNX2, SIX4_A, STX16, TAL1, TMEM30B, and TSPAN33 (see, Tables 4, 5A, and 5B). 
     In some embodiments wherein the sample obtained from the subject is skin tissue and the methylation state of one or more of the following markers is different than a methylation state of the one or more markers assayed in a subject that does not have melanoma indicates the subject has melanoma: MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-73073830, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22541874-22541948, ME3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763 (see, Tables 9, 10 and 11). 
     In some embodiments wherein the sample obtained from the subject is skin tissue and the methylation state of one or more of the following markers is different than a methylation state of the one or more markers assayed in a subject that does not have melanoma indicates the subject has melanoma: AGRN, BMP8B, c17orf46_B, c17orf57_B, C2CD4A, C2CD4D_B, CDYL, CMAH, DENND2D, DLEU2, DSCR6, FLJ22536_B, FLJ45983, FOXF2, GALNT3_B, HCG4P6, HOXA9_B, KIFC2, LDLRAD2, LY75, LYL1, LYN, MAPK13, MAX.chr1.1072486-1072508, MAX.chr1.32237693-32237785, MAX.chr10.62492374-62492793, MAX.chr11.14926535-14926715, MAX.chr16.54970444-54970469, MAX.chr19.16439332-16439390, MAX.chr20.21080670-21081280, MAX.chr4.113432264-113432298, MAX.chr7.129425668-129425719, MAX.chr8.82543163-82543213, PARP15, PRKAG2, PROC_A, PROM1_B, PTGER4_A, PTP4A3, SDCCAG8, SH3PXD2A, SLC2A2, SLC35D3, TBR1, TBX2, TCP11, TFAP2A, and TRIM73 (see, Tables 7A and 7B). 
     In some embodiments wherein the sample obtained from the subject is skin tissue and the methylation state of one or more of the following markers is different than a methylation state of the one or more markers assayed in a subject that does not have metastatic melanoma indicates the subject has metastatic melanoma: MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763, FOXL2NB, and HOXA9_A (see, Table 5C, Example I). 
     In some embodiments wherein the sample obtained from the subject is skin tissue and the methylation state of one or more of the following markers is different than a methylation state of the one or more markers assayed in a subject that does not have metastatic melanoma indicates the subject has metastatic melanoma: MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-73073830, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22541874-22541948, ME3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763 (see, Tables 9, 10 and 11). 
     In some embodiments wherein the sample obtained from the subject is skin tissue and the methylation state of one or more of the following markers is different than a methylation state of the one or more markers assayed in a subject that does not have primary cutaneous melanoma indicates the subject has primary cutaneous melanoma: MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763, FOXL2NB, and HOXA9_A (see, Table 5C, Example I). 
     In some embodiments wherein the sample obtained from the subject is skin tissue and the methylation state of one or more of the following markers is different than a methylation state of the one or more markers assayed in a subject that does not have primary cutaneous melanoma indicates the subject has primary cutaneous melanoma: MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-73073830, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22541874-22541948, ME3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763 (see, Tables 9, 10 and 11). 
     In some embodiments wherein the sample obtained from the subject is a blood sample (e.g., plasma sample, whole blood sample, leukocyte sample, serum sample) and the methylation state of one or more of the following markers is different than a methylation state of the one or more markers assayed in a subject that does not have melanoma indicates the subject has melanoma: c10orf55, c2orf82, FOXL2NB, CLIC5, FAM174B_B, FLJ22536_A, FOXP1, GALNT3_A, HOPX, HOXA9_A, ITPKA, KCNQ4_A, LMX1B, MAX.chr1.110627096-110627364, MAX.chr10.22541869-22541953, MAX.chr10.62492690-62492812, MAX.chr13.29106812-29106917, MAX.chr17.73073682-73073830, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, MAX.chr5.42992655-42992768, MAX.chr5.60921627-60921853, MAX.chr6.29521537-29521696, MAX.chr7.155259597-155259763, ME3, MIR155HG, NFATC2_A, OCA2, OXT, RNF207_A, RUNX2, SIX4_A, STX16, TAL1, TMEM30B, and TSPAN33 (see, Table 4, 5A, 5B, 6; Example I). 
     In some embodiments wherein the sample obtained from the subject is a blood sample (e.g., plasma sample, whole blood sample, leukocyte sample, serum sample) and the methylation state of one or more of the following markers is different than a methylation state of the one or more markers assayed in a subject that does not have melanoma indicates the subject has melanoma: MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-73073830, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22541874-22541948, ME3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763 (see, Tables 9, 10 and 11). 
     In some embodiments wherein the sample obtained from the subject is a blood sample (e.g., plasma sample, whole blood sample, leukocyte sample, serum sample) and the methylation state of one or more of the following markers is different than a methylation state of the one or more markers assayed in a subject that does not have melanoma indicates the subject has melanoma: one or more of the markers recited in Table 8A. 
     The technology is related to identifying and discriminating melanoma and/or various forms of melanoma (e.g., metastatic melanoma, primary cutaneous melanoma). Some embodiments provide methods comprising assaying a plurality of markers, e.g., comprising assaying 1, 2, 3, 2 to 11 to 100 or 120 or 331 markers (e.g., 1-4, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18, 1-19, 1-20, 1-25, 1-50, 1-75, 1-100, 1-200, 1-300, 1-331) (e.g., 2-4, 2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 2-25, 2-50, 2-75, 2-100, 2-200, 2-300, 2-331) (e.g., 3-4, 3-6, 3-7, 3-8, 3-9, 3-10, 3-11, 3-12, 3-13, 3-14, 3-15, 3-16, 3-17, 3-18, 3-19, 3-20, 3-25, 3-50, 3-75, 3-100, 3-200, 3-300, 3-331) (e.g., 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 4-12, 4-13, 4-14, 4-15, 4-16, 4-17, 4-18, 4-19, 4-20, 4-25, 4-50, 4-75, 4-100, 4-200, 4-300, 4-331) (e.g., 5-6, 5-7, 5-8, 5-9, 5-10, 5-11, 5-12, 5-13, 5-14, 5-15, 5-16, 5-17, 5-18, 5-19, 5-20, 5-25, 5-50, 5-75, 5-100, 5-200, 5-300, 5-331). 
     The technology is not limited in the methylation state assessed. In some embodiments assessing the methylation state of the marker in the sample comprises determining the methylation state of one base. In some embodiments, assaying the methylation state of the marker in the sample comprises determining the extent of methylation at a plurality of bases. Moreover, in some embodiments the methylation state of the marker comprises an increased methylation of the marker relative to a normal methylation state of the marker. In some embodiments, the methylation state of the marker comprises a decreased methylation of the marker relative to a normal methylation state of the marker. In some embodiments the methylation state of the marker comprises a different pattern of methylation of the marker relative to a normal methylation state of the marker. 
     Furthermore, in some embodiments the marker is a region of 100 or fewer bases, the marker is a region of 500 or fewer bases, the marker is a region of 1000 or fewer bases, the marker is a region of 5000 or fewer bases, or, in some embodiments, the marker is one base. In some embodiments the marker is in a high CpG density promoter. 
     The technology is not limited by sample type. For example, in some embodiments the sample is a stool sample, a tissue sample (e.g., skin tissue sample), lymphatic tissue, deep tissue biopsy, a blood sample (e.g., plasma, serum, whole blood), an excretion, or a urine sample. In some embodiments, the sample is a fine needle aspirate. In some embodiments, the sample is taken by using a sampling device such as a swab or tape with adhesive to collect cells on the skin surface. Malignant melanoma is characterized by spread to other organs and deep tissue types, especially to lymph nodes, and to other organs such as the lungs, liver, bone or brain. 
     Furthermore, the technology is not limited in the method used to determine methylation state. In some embodiments the assaying comprises using methylation specific polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation specific nuclease, mass-based separation, or target capture. In some embodiments, the assaying comprises use of a methylation specific oligonucleotide. In some embodiments, the technology uses massively parallel sequencing (e.g., next-generation sequencing) to determine methylation state, e.g., sequencing-by-synthesis, real-time (e.g., single-molecule) sequencing, bead emulsion sequencing, nanopore sequencing, etc. 
     The technology provides reagents for detecting a DMR, e.g., in some embodiments are provided a set of oligonucleotides comprising the sequences provided by SEQ ID NO: 1-167 (see, Table 3, 10 and 11). In some embodiments are provided an oligonucleotide comprising a sequence complementary to a chromosomal region having a base in a DMR, e.g., an oligonucleotide sensitive to methylation state of a DMR. 
     The technology provides various panels of markers use for identifying melanoma, e.g., in some embodiments the marker comprises a chromosomal region having an annotation that is c10orf55, c2orf82, FOXL2NB, CLIC5, FAM174B_B, FLJ22536_A, FOXP1, GALNT3_A, HOPX, HOXA9_A, ITPKA, KCNQ4_A, LMX1B, MAX.chr1.110627096-110627364, MAX.chr10.22541869-22541953, MAX.chr10.62492690-62492812, MAX.chr13.29106812-29106917, MAX.chr17.73073682-73073830, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, MAX.chr5.42992655-42992768, MAX.chr5.60921627-60921853, MAX.chr6.29521537-29521696, MAX.chr7.155259597-155259763, ME3, MIR155HG, NFATC2_A, OCA2, OXT, RNF207_A, RUNX2, SIX4_A, STX16, TAL1, TMEM30B, and TSPAN33 (see, Tables 4, 5A, 5B). 
     The technology provides various panels of markers use for identifying melanoma, e.g., in some embodiments the marker comprises a chromosomal region having an annotation that is MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-73073830, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22541874-22541948, ME3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763 (see, Tables 9, 10 and 11). 
     The technology provides various panels of markers use for identifying melanoma, e.g., in some embodiments the marker comprises a chromosomal region having an annotation that is AGRN, BMP8B, c17orf46_B, c17orf57_B, C2CD4A, C2CD4D_B, CDYL, CMAH, DENND2D, DLEU2, DSCR6, FLJ22536_B, FLJ45983, FOXF2, GALNT3_B, HCG4P6, HOXA9_B, KIFC2, LDLRAD2, LY75, LYL1, LYN, MAPK13, MAX.chr1.1072486-1072508, MAX.chr1.32237693-32237785, MAX.chr10.62492374-62492793, MAX.chr11.14926535-14926715, MAX.chr16.54970444-54970469, MAX.chr19.16439332-16439390, MAX.chr20.21080670-21081280, MAX.chr4.113432264-113432298, MAX.chr7.129425668-129425719, MAX.chr8.82543163-82543213, PARP15, PRKAG2, PROC_A, PROM1_B, PTGER4_A, PTP4A3, SDCCAG8, SH3PXD2A, SLC2A2, SLC35D3, TBR1, TBX2, TCP11, TFAP2A, and TRIM73 (see, Tables 7A and 7B). 
     The technology provides various panels of markers use for identifying metastatic melanoma, e.g., in some embodiments the marker comprises a chromosomal region having an annotation that is MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763, FOXL2NB, and HOXA9_A (see, Table 5C, Example I). 
     The technology provides various panels of markers use for identifying metastatic melanoma, e.g., in some embodiments the marker comprises a chromosomal region having an annotation that is MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-73073830, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22541874-22541948, ME3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763 (see, Tables 9, 10 and 11). 
     The technology provides various panels of markers use for identifying primary cutaneous melanoma, e.g., in some embodiments the marker comprises a chromosomal region having an annotation that is MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763, FOXL2NB, and HOXA9_A (see, Table 5C, Example I). 
     The technology provides various panels of markers use for identifying primary cutaneous melanoma, e.g., in some embodiments the marker comprises a chromosomal region having an annotation that is MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-73073830, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22541874-22541948, ME3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763 (see, Tables 9, 10 and 11). 
     The technology provides various panels of markers use for identifying melanoma, e.g., in some embodiments the marker comprises a chromosomal region having an annotation that is recited in Table 8A (see, Example II). 
     Kit embodiments are provided, e.g., a kit comprising a reagent capable of modifying DNA in a methylation-specific manner (e.g., a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, Ten Eleven Translocation (TET) enzyme (e.g., human TET1, human TET2, human TET3, murine TET1, murine TET2, murine TET3,  Naegleria  TET (NgTET),  Coprinopsis cinerea  (CcTET)), or a variant thereof), organic borane); and a control nucleic acid comprising one or more sequences from DMR 1-331 (from Tables 1A, 2A, 7A, 8A, and 9) and having a methylation state associated with a subject who does not have cancer. In some embodiments, kits comprise a bisulfite reagent and an oligonucleotide as described herein. In some embodiments, kits comprise a reagent capable of modifying DNA in a methylation-specific manner (e.g., a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, Ten Eleven Translocation (TET) enzyme (e.g., human TET1, human TET2, human TET3, murine TET1, murine TET2, murine TET3,  Naegleria  TET (NgTET),  Coprinopsis cinerea  (CcTET)), or a variant thereof), organic borane); and a control nucleic acid comprising one or more sequences from DMR 1-331 (from Tables 1A, 2A, 7A, 8A, and 9) and having a methylation state associated with a subject who has a specific type of cancer. Some kit embodiments comprise a sample collector for obtaining a sample from a subject (e.g., a stool sample; tissue sample; plasma sample, serum sample, whole blood sample); a reagent capable of modifying DNA in a methylation-specific manner (e.g., a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, Ten Eleven Translocation (TET) enzyme (e.g., human TET1, human TET2, human TET3, murine TET1, murine TET2, murine TET3,  Naegleria  TET (NgTET),  Coprinopsis cinerea  (CcTET)), or a variant thereof), organic borane); and an oligonucleotide as described herein. 
     The technology is related to embodiments of compositions (e.g., reaction mixtures). In some embodiments are provided a composition comprising a nucleic acid comprising a DMR and a reagent capable of modifying DNA in a methylation-specific manner (e.g., a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, Ten Eleven Translocation (TET) enzyme (e.g., human TET1, human TET2, human TET3, murine TET1, murine TET2, murine TET3,  Naegleria  TET (NgTET),  Coprinopsis cinerea  (CcTET)), or a variant thereof), organic borane). Some embodiments provide a composition comprising a nucleic acid comprising a DMR and an oligonucleotide as described herein. Some embodiments provide a composition comprising a nucleic acid comprising a DMR and a methylation-sensitive restriction enzyme. Some embodiments provide a composition comprising a nucleic acid comprising a DMR and a polymerase. 
     Additional related method embodiments are provided for screening for melanoma and/or various forms of melanoma (e.g., metastatic melanoma, primary cutaneous melanoma) in a sample obtained from a subject (e.g., a stool sample; skin tissue sample; fine needle aspirate, deep tissue sample, plasma sample, serum sample, whole blood sample), e.g., a method comprising determining a methylation state of a marker in the sample comprising a base in a DMR that is one or more of DMR 1-331 (from Tables 1A, 2A, 7A, 8A, and 9); comparing the methylation state of the marker from the subject sample to a methylation state of the marker from a normal control sample from a subject who does not have melanoma (e.g., melanoma and/or a form of melanoma: metastatic melanoma, primary cutaneous melanoma); and determining a confidence interval and/or a p value of the difference in the methylation state of the subject sample and the normal control sample. In some embodiments, the confidence interval is 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% or 99.99% and the p value is 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, or 0.0001. Some embodiments of methods provide steps of reacting a nucleic acid comprising a DMR with a reagent capable of modifying nucleic acid in a methylation-specific manner (e.g., a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, Ten Eleven Translocation (TET) enzyme (e.g., human TET1, human TET2, human TET3, murine TET1, murine TET2, murine TET3,  Naegleria  TET (NgTET),  Coprinopsis cinerea  (CcTET)), or a variant thereof), organic borane) to produce, for example, nucleic acid modified in a methylation-specific manner; sequencing the nucleic acid modified in a methylation-specific manner to provide a nucleotide sequence of the nucleic acid modified in a methylation-specific manner; comparing the nucleotide sequence of the nucleic acid modified in a methylation-specific manner with a nucleotide sequence of a nucleic acid comprising the DMR from a subject who does not have a specific type of cancer to identify differences in the two sequences; and identifying the subject as having melanoma and/or a form of melanoma (e.g., metastatic melanoma, primary cutaneous melanoma) when a difference is present. 
     Systems for screening for melanoma in a sample obtained from a subject are provided by the technology. Exemplary embodiments of systems include, e.g., a system for screening for melanoma and/or types of melanoma (e.g., metastatic melanoma, primary cutaneous melanoma) in a sample obtained from a subject (e.g., a stool sample; skin tissue sample; fine needle aspirate, deep tissue sample, plasma sample, serum sample, whole blood sample), the system comprising an analysis component configured to determine the methylation state of a sample, a software component configured to compare the methylation state of the sample with a control sample or a reference sample methylation state recorded in a database, and an alert component configured to alert a user of a melanoma-associated methylation state. An alert is determined in some embodiments by a software component that receives the results from multiple assays (e.g., determining the methylation states of multiple markers, e.g., DMR, e.g., as provided in Tables 1A, 2A, 7A, 8A, and 9) and calculating a value or result to report based on the multiple results. Some embodiments provide a database of weighted parameters associated with each DMR provided herein for use in calculating a value or result and/or an alert to report to a user (e.g., such as a physician, nurse, clinician, etc.). In some embodiments all results from multiple assays are reported and in some embodiments one or more results are used to provide a score, value, or result based on a composite of one or more results from multiple assays that is indicative of a cancer risk in a subject. 
     In some embodiments of systems, a sample comprises a nucleic acid comprising a DMR. In some embodiments the system further comprises a component for isolating a nucleic acid, a component for collecting a sample such as a component for collecting a stool sample. In some embodiments, the system comprises nucleic acid sequences comprising a DMR. In some embodiments the database comprises nucleic acid sequences from subjects who do not have melanoma and/or specific types of melanoma (e.g., metastatic melanoma, primary cutaneous melanoma). Also provided are nucleic acids, e.g., a set of nucleic acids, each nucleic acid having a sequence comprising a DMR. In some embodiments the set of nucleic acids wherein each nucleic acid has a sequence from a subject who does not have melanoma and/or specific types of melanoma. Related system embodiments comprise a set of nucleic acids as described and a database of nucleic acid sequences associated with the set of nucleic acids. Some embodiments further comprise a reagent capable of modifying DNA in a methylation-specific manner (e.g., a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, Ten Eleven Translocation (TET) enzyme (e.g., human TET1, human TET2, human TET3, murine TET1, murine TET2, murine TET3,  Naegleria  TET (NgTET),  Coprinopsis cinerea  (CcTET)), or a variant thereof), organic borane). Some embodiments further comprise a nucleic acid sequencer. 
     In certain embodiments, methods for characterizing a sample (e.g., a stool sample; skin tissue sample; fine needle aspirate, deep tissue sample, plasma sample, serum sample, whole blood sample) from a human patient are provided. For example, in some embodiments such embodiments comprise obtaining DNA from a sample of a human patient; assaying a methylation state of a DNA methylation marker comprising a base in a differentially methylated region (DMR) selected from a group consisting of DMR 1-331 from Tables TA, 2A, 7A, 8A, and 9; and comparing the assayed methylation state of the one or more DNA methylation markers with methylation level references for the one or more DNA methylation markers for human patients not having melanoma and/or specific types of melanoma (e.g., metastatic melanoma, primary cutaneous melanoma). 
     Such methods are not limited to a particular type of sample from a human patient. In some embodiments, the sample is a skin tissue sample. In some embodiments, the sample is a plasma sample. In some embodiments, the sample is a stool sample, a tissue sample, a skin tissue sample, a blood sample (e.g., a stool sample; skin tissue sample; fine needle aspirate, deep tissue sample, plasma sample, serum sample, whole blood sample). 
     In some embodiments, such methods comprise assaying a plurality of DNA methylation markers (e.g., 1-4, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18, 1-19, 1-20, 1-25, 1-50, 1-75, 1-100, 1-200, 1-300, 1-331) (e.g., 2-4, 2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 2-25, 2-50, 2-75, 2-100, 2-200, 2-300, 2-331) (e.g., 3-4, 3-6, 3-7, 3-8, 3-9, 3-10, 3-11, 3-12, 3-13, 3-14, 3-15, 3-16, 3-17, 3-18, 3-19, 3-20, 3-25, 3-50, 3-75, 3-100, 3-200, 3-300, 3-331) (e.g., 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 4-12, 4-13, 4-14, 4-15, 4-16, 4-17, 4-18, 4-19, 4-20, 4-25, 4-50, 4-75, 4-100, 4-200, 4-300, 4-331) (e.g., 5-6, 5-7, 5-8, 5-9, 5-10, 5-11, 5-12, 5-13, 5-14, 5-15, 5-16, 5-17, 5-18, 5-19, 5-20, 5-25, 5-50, 5-75, 5-100, 5-200, 5-300, 5-331). In some embodiments, such methods comprise assaying 2 to 11 DNA methylation markers. In some embodiments, such methods comprise assaying 12 to 120 DNA methylation markers. In some embodiments, such methods comprise assaying 2 to 331 DNA methylation markers. In some embodiments, such methods comprise assaying the methylation state of the one or more DNA methylation markers in the sample comprises determining the methylation state of one base. In some embodiments, such methods comprise assaying the methylation state of the one or more DNA methylation markers in the sample comprises determining the extent of methylation at a plurality of bases. In some embodiments, such methods comprise assaying a methylation state of a forward strand or assaying a methylation state of a reverse strand. 
     In some embodiments, the DNA methylation marker is a region of 100 or fewer bases. In some embodiments, the DNA methylation marker is a region of 500 or fewer bases. In some embodiments, the DNA methylation marker is a region of 1000 or fewer bases. In some embodiments, the DNA methylation marker is a region of 5000 or fewer bases. In some embodiments, the DNA methylation marker is one base. In some embodiments, the DNA methylation marker is in a high CpG density promoter. 
     In some embodiments, the assaying comprises using methylation specific polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation specific nuclease, mass-based separation, or target capture. 
     In some embodiments, the assaying comprises use of a methylation specific oligonucleotide. In some embodiments, the methylation specific oligonucleotide is selected from the group consisting of SEQ ID NO: 1-80 (Table 3). 
     In some embodiments, a chromosomal region having an annotation selected from the group consisting of c10orf55, c2orf82, FOXL2NB, CLIC5, FAM174B_B, FLJ22536_A, FOXP1, GALNT3_A, HOPX, HOXA9_A, ITPKA, KCNQ4_A, LMX1B, MAX.chr1.110627096-110627364, MAX.chr10.22541869-22541953, MAX.chr10.62492690-62492812, MAX.chr13.29106812-29106917, MAX.chr17.73073682-73073830, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, MAX.chr5.42992655-42992768, MAX.chr5.60921627-60921853, MAX.chr6.29521537-29521696, MAX.chr7.155259597-155259763, ME3, MIR155HG, NFATC2_A, OCA2, OXT, RNF207_A, RUNX2, SIX4_A, STX16, TAL1, TMEM30B, and TSPAN33 (see, Tables 4, 5A, 5B and 6) comprises the DNA methylation marker. 
     In some embodiments, a chromosomal region having an annotation selected from the group consisting of AGRN, BMP8B, c17orf46_B, c17orf57_B, C2CD4A, C2CD4D_B, CDYL, CMAH, DENND2D, DLEU2, DSCR6, FLJ22536_B, FLJ45983, FOXF2, GALNT3_B, HCG4P6, HOXA9_B, KIFC2, LDLRAD2, LY75, LYL1, LYN, MAPK13, MAX.chr1.1072486-1072508, MAX.chr1.32237693-32237785, MAX.chr10.62492374-62492793, MAX.chr11.14926535-14926715, MAX.chr16.54970444-54970469, MAX.chr19.16439332-16439390, MAX.chr20.21080670-21081280, MAX.chr4.113432264-113432298, MAX.chr7.129425668-129425719, MAX.chr8.82543163-82543213, PARP15, PRKAG2, PROC_A, PROM1_B, PTGER4_A, PTP4A3, SDCCAG8, SH3PXD2A, SLC2A2, SLC35D3, TBR1, TBX2, TCP11, TFAP2A, and TRIM73 (see, Tables 7A and 7B) comprises the DNA methylation marker. 
     In some embodiments, a chromosomal region having an annotation selected from the group consisting of MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-73073830, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22541874-22541948, ME3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763 (see, Tables 9, 10 and 11) comprises the DNA methylation marker. 
     In some embodiments, a chromosomal region having an annotation recited in Table 8A (see, Example II) comprises the DNA methylation marker. 
     In some embodiments, a chromosomal region having an annotation selected from the group consisting of MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763, FOXL2NB, and HOXA9_A (see, Table 5C, Example I) comprises the DNA methylation marker. 
     In some embodiments, such methods comprise determining the methylation state of two DNA methylation markers. In some embodiments, such methods comprise determining the methylation state of a pair of DNA methylation markers provided in Tables 1A, 2A, 7A 8A, and/or 9. 
     In certain embodiments, the technology provides methods for characterizing a sample (e.g., a stool sample; skin tissue sample; fine needle aspirate, deep tissue sample, plasma sample, serum sample, whole blood sample) obtained from a human patient. In some embodiments, such methods comprise determining a methylation state of a DNA methylation marker in the sample comprising a base in a DMR selected from a group consisting of DMR 1-331 from Tables 1A, 2A, 7A, 8A, and 9; comparing the methylation state of the DNA methylation marker from the patient sample to a methylation state of the DNA methylation marker from a normal control sample from a human subject who does not have a melanoma and/or a specific form of melanoma (e.g., metastatic melanoma, primary cutaneous melanoma); and determining a confidence interval and/or a p value of the difference in the methylation state of the human patient and the normal control sample. In some embodiments, the confidence interval is 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% or 99.99% and the p value is 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, or 0.0001. 
     In certain embodiments, the technology provides methods for characterizing a sample obtained from a human subject (e.g., a stool sample; skin tissue sample; fine needle aspirate, deep tissue sample, plasma sample, serum sample, whole blood sample), the method comprising reacting a nucleic acid comprising a DMR with a reagent capable of modifying DNA in a methylation-specific manner (e.g., a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and a bisulfite reagent) to produce nucleic acid modified in a methylation-specific manner; sequencing the nucleic acid modified in a methylation-specific manner to provide a nucleotide sequence of the nucleic acid modified in a methylation-specific manner; comparing the nucleotide sequence of the nucleic acid modified in a methylation-specific manner with a nucleotide sequence of a nucleic acid comprising the DMR from a subject who does not have melanoma to identify differences in the two sequences. 
     In certain embodiments, the technology provides systems for characterizing a sample obtained from a human subject (e.g., a stool sample; skin tissue sample; fine needle aspirate, deep tissue sample, plasma sample, serum sample, whole blood sample), the system comprising an analysis component configured to determine the methylation state of a sample, a software component configured to compare the methylation state of the sample with a control sample or a reference sample methylation state recorded in a database, and an alert component configured to determine a single value based on a combination of methylation states and alert a user of a melanoma-associated methylation state. In some embodiments, the sample comprises a nucleic acid comprising a DMR. 
     In some embodiments, such systems further comprise a component for isolating a nucleic acid. In some embodiments, such systems further comprise a component for collecting a sample. 
     In some embodiments, the sample is a stool sample, a tissue sample, a skin tissue sample, fine needle aspirate, a deep tissue sample, a lymph node sample, a blood sample (e.g., plasma sample, whole blood sample, serum sample), or a urine sample. 
     In some embodiments, the database comprises nucleic acid sequences comprising a DMR. In some embodiments, the database comprises nucleic acid sequences from subjects who do not have a melanoma. 
     In some embodiments, the sample is a stool sample, a tissue sample (e.g., skin tissue), a blood sample (e.g., plasma sample, whole blood sample, serum sample), or a urine sample. In some embodiments, the sample comprises blood, serum, plasma, gastric secretions, pancreatic juice, a cerebral spinal fluid (CSF) sample, a gastrointestinal biopsy sample, and/or cells recovered from stool. In some embodiments, the subject is human. The sample may include cells, secretions, or tissues from the lymph gland, breast, liver, bile ducts, pancreas, stomach, colon, rectum, esophagus, small intestine, appendix, duodenum, polyps, gall bladder, anus, and/or peritoneum. In some embodiments, the sample comprises cellular fluid, ascites, urine, feces, gastric section, pancreatic fluid, fluid obtained during endoscopy, blood. 
     Additional embodiments will be apparent to persons skilled in the relevant art based on the teachings contained herein. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG.  1   : Marker chromosomal regions used for the methylated DNA markers recited in Table 9 and related primer and probe information (Tables 10 and 11). 
     
    
    
     DEFINITIONS 
     To facilitate an understanding of the present technology, a number of terms and phrases are defined below. Additional definitions are set forth throughout the detailed description. 
     Throughout the specification and claims, the following terms take the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrase “in one embodiment” as used herein does not necessarily refer to the same embodiment, though it may. Furthermore, the phrase “in another embodiment” as used herein does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the invention. 
     In addition, as used herein, the term “or” is an inclusive “or” operator and is equivalent to the term “and/or” unless the context clearly dictates otherwise. The term “based on” is not exclusive and allows for being based on additional factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meaning of “a”, “an”, and “the” include plural references. The meaning of “in” includes “in” and “on.” 
     The transitional phrase “consisting essentially of” as used in claims in the present application limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention, as discussed in In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976). For example, a composition “consisting essentially of” recited elements may contain an unrecited contaminant at a level such that, though present, the contaminant does not alter the function of the recited composition as compared to a pure composition, i.e., a composition “consisting of” the recited components. 
     As used herein, a “nucleic acid” or “nucleic acid molecule” generally refers to any ribonucleic acid or deoxyribonucleic acid, which may be unmodified or modified DNA or RNA. “Nucleic acids” include, without limitation, single- and double-stranded nucleic acids. As used herein, the term “nucleic acid” also includes DNA as described above that contains one or more modified bases. Thus, DNA with a backbone modified for stability or for other reasons is a “nucleic acid”. The term “nucleic acid” as it is used herein embraces such chemically, enzymatically, or metabolically modified forms of nucleic acids, as well as the chemical forms of DNA characteristic of viruses and cells, including for example, simple and complex cells. 
     The terms “oligonucleotide” or “polynucleotide” or “nucleotide” or “nucleic acid” refer to a molecule having two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and usually more than ten. The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide. The oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof. Typical deoxyribonucleotides for DNA are thymine, adenine, cytosine, and guanine. Typical ribonucleotides for RNA are uracil, adenine, cytosine, and guanine. 
     As used herein, the terms “locus” or “region” of a nucleic acid refer to a subregion of a nucleic acid, e.g., a gene on a chromosome, a single nucleotide, a CpG island, etc. 
     The terms “complementary” and “complementarity” refer to nucleotides (e.g., 1 nucleotide) or polynucleotides (e.g., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence 5′-A-G-T-3′ is complementary to the sequence 3′-T-C-A-5′. Complementarity may be “partial,” in which only some of the nucleic acids&#39; bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands effects the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions and in detection methods that depend upon binding between nucleic acids. 
     The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of an RNA, or of a polypeptide or its precursor. A functional polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence as long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, etc.) of the polypeptide are retained. The term “portion” when used in reference to a gene refers to fragments of that gene. The fragments may range in size from a few nucleotides to the entire gene sequence minus one nucleotide. Thus, “a nucleotide comprising at least a portion of a gene” may comprise fragments of the gene or the entire gene. 
     The term “gene” also encompasses the coding regions of a structural gene and includes sequences located adjacent to the coding region on both the 5′ and 3′ ends, e.g., for a distance of about 1 kb on either end, such that the gene corresponds to the length of the full-length mRNA (e.g., comprising coding, regulatory, structural and other sequences). The sequences that are located 5′ of the coding region and that are present on the mRNA are referred to as 5′ non-translated or untranslated sequences. The sequences that are located 3′ or downstream of the coding region and that are present on the mRNA are referred to as 3′ non-translated or 3′ untranslated sequences. The term “gene” encompasses both cDNA and genomic forms of a gene. In some organisms (e.g., eukaryotes), a genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed “introns” or “intervening regions” or “intervening sequences.” Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide. 
     In addition to containing introns, genomic forms of a gene may also include sequences located on both the 5′ and 3′ ends of the sequences that are present on the RNA transcript. These sequences are referred to as “flanking” sequences or regions (these flanking sequences are located 5′ or 3′ to the non-translated sequences present on the mRNA transcript). The 5′ flanking region may contain regulatory sequences such as promoters and enhancers that control or influence the transcription of the gene. The 3′ flanking region may contain sequences that direct the termination of transcription, posttranscriptional cleavage, and poly adenylation. 
     The term “wild-type” when made in reference to a gene refers to a gene that has the characteristics of a gene isolated from a naturally occurring source. The term “wild-type” when made in reference to a gene product refers to a gene product that has the characteristics of a gene product isolated from a naturally occurring source. The term “naturally-occurring” as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by the hand of a person in the laboratory is naturally-occurring. A wild-type gene is often that gene or allele that is most frequently observed in a population and is thus arbitrarily designated the “normal” or “wild-type” form of the gene. In contrast, the term “modified” or “mutant” when made in reference to a gene or to a gene product refers, respectively, to a gene or to a gene product that displays modifications in sequence and/or functional properties (e.g., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product. 
     The term “allele” refers to a variation of a gene; the variations include but are not limited to variants and mutants, polymorphic loci, and single nucleotide polymorphic loci, frameshift, and splice mutations. An allele may occur naturally in a population or it might arise during the lifetime of any particular individual of the population. 
     Thus, the terms “variant” and “mutant” when used in reference to a nucleotide sequence refer to a nucleic acid sequence that differs by one or more nucleotides from another, usually related, nucleotide acid sequence. A “variation” is a difference between two different nucleotide sequences; typically, one sequence is a reference sequence. 
     “Amplification” is a special case of nucleic acid replication involving template specificity. It is to be contrasted with non-specific template replication (e.g., replication that is template-dependent but not dependent on a specific template). Template specificity is here distinguished from fidelity of replication (e.g., synthesis of the proper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-) specificity. Template specificity is frequently described in terms of “target” specificity. Target sequences are “targets” in the sense that they are sought to be sorted out from other nucleic acid. Amplification techniques have been designed primarily for this sorting out. 
     The term “amplifying” or “amplification” in the context of nucleic acids refers to the production of multiple copies of a polynucleotide, or a portion of the polynucleotide, typically starting from a small amount of the polynucleotide (e.g., a single polynucleotide molecule), where the amplification products or amplicons are generally detectable. Amplification of polynucleotides encompasses a variety of chemical and enzymatic processes. The generation of multiple DNA copies from one or a few copies of a target or template DNA molecule during a polymerase chain reaction (PCR) or a ligase chain reaction (LCR; see, e.g., U.S. Pat. No. 5,494,810; herein incorporated by reference in its entirety) are forms of amplification. Additional types of amplification include, but are not limited to, allele-specific PCR (see, e.g., U.S. Pat. No. 5,639,611; herein incorporated by reference in its entirety), assembly PCR (see, e.g., U.S. Pat. No. 5,965,408; herein incorporated by reference in its entirety), helicase-dependent amplification (see, e.g., U.S. Pat. No. 7,662,594; herein incorporated by reference in its entirety), hot-start PCR (see, e.g., U.S. Pat. Nos. 5,773,258 and 5,338,671; each herein incorporated by reference in their entireties), intersequence-specific PCR, inverse PCR (see, e.g., Triglia, et al. (1988) Nucleic Acids Res., 16:8186; herein incorporated by reference in its entirety), ligation-mediated PCR (see, e.g., Guilfoyle, R. et al., Nucleic Acids Research, 25:1854-1858 (1997); U.S. Pat. No. 5,508,169; each of which are herein incorporated by reference in their entireties), methylation-specific PCR (see, e.g., Herman, et al., (1996) PNAS 93(13) 9821-9826; herein incorporated by reference in its entirety), miniprimer PCR, multiplex ligation-dependent probe amplification (see, e.g., Schouten, et al., (2002) Nucleic Acids Research 30(12): e57; herein incorporated by reference in its entirety), multiplex PCR (see, e.g., Chamberlain, et al., (1988) Nucleic Acids Research 16(23) 11141-11156; Ballabio, et al., (1990) Human Genetics 84(6) 571-573; Hayden, et al., (2008) BMC Genetics 9:80; each of which are herein incorporated by reference in their entireties), nested PCR, overlap-extension PCR (see, e.g., Higuchi, et al., (1988) Nucleic Acids Research 16(15) 7351-7367; herein incorporated by reference in its entirety), real time PCR (see, e.g., Higuchi, et al., (1992) Biotechnology 10:413-417; Higuchi, et al., (1993) Biotechnology 11:1026-1030; each of which are herein incorporated by reference in their entireties), reverse transcription PCR (see, e.g., Bustin, S. A. (2000) J. Molecular Endocrinology 25:169-193; herein incorporated by reference in its entirety), solid phase PCR, thermal asymmetric interlaced PCR, and Touchdown PCR (see, e.g., Don, et al., Nucleic Acids Research (1991) 19(14) 4008; Roux, K. (1994) Biotechniques 16(5) 812-814; Hecker, et al., (1996) Biotechniques 20(3) 478-485; each of which are herein incorporated by reference in their entireties). Polynucleotide amplification also can be accomplished using digital PCR (see, e.g., Kalinina, et al., Nucleic Acids Research. 25; 1999-2004, (1997); Vogelstein and Kinzler, Proc Natl Acad Sci USA. 96; 9236-41, (1999); International Patent Publication No. WO05023091A2; US Patent Application Publication No. 20070202525; each of which are incorporated herein by reference in their entireties). 
     The term “polymerase chain reaction” (“PCR”) refers to the method of K. B. Mullis U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188, that describe a method for increasing the concentration of a segment of a target sequence in a mixture of genomic or other DNA or RNA, without cloning or purification. This process for amplifying the target sequence consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the double stranded target sequence. To effect amplification, the mixture is denatured and the primers then annealed to their complementary sequences within the target molecule. Following annealing, the primers are extended with a polymerase so as to form a new pair of complementary strands. The steps of denaturation, primer annealing, and polymerase extension can be repeated many times (i.e., denaturation, annealing and extension constitute one “cycle”; there can be numerous “cycles”) to obtain a high concentration of an amplified segment of the desired target sequence. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of the repeating aspect of the process, the method is referred to as the “polymerase chain reaction” (“PCR”). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be “PCR amplified” and are “PCR products” or “amplicons.” Those of skill in the art will understand the term “PCR” encompasses many variants of the originally described method using, e.g., real time PCR, nested PCR, reverse transcription PCR (RT-PCR), single primer and arbitrarily primed PCR, etc. 
     Template specificity is achieved in most amplification techniques by the choice of enzyme. Amplification enzymes are enzymes that, under conditions they are used, will process only specific sequences of nucleic acid in a heterogeneous mixture of nucleic acid. For example, in the case of Q-beta replicase, MDV-1 RNA is the specific template for the replicase (Kacian et al., Proc. Natl. Acad. Sci. USA, 69:3038 [1972]). Other nucleic acid will not be replicated by this amplification enzyme. Similarly, in the case of T7 RNA polymerase, this amplification enzyme has a stringent specificity for its own promoters (Chamberlin et al, Nature, 228:227 [1970]). In the case of T4 DNA ligase, the enzyme will not ligate the two oligonucleotides or polynucleotides, where there is a mismatch between the oligonucleotide or polynucleotide substrate and the template at the ligation junction (Wu and Wallace (1989) Genomics 4:560). Finally, thermostable template-dependant DNA polymerases (e.g., Taq and Pfu DNA polymerases), by virtue of their ability to function at high temperature, are found to display high specificity for the sequences bounded and thus defined by the primers; the high temperature results in thermodynamic conditions that favor primer hybridization with the target sequences and not hybridization with non-target sequences (H. A. Erlich (ed.), PCR Technology, Stockton Press [1989]). 
     As used herein, the term “nucleic acid detection assay” refers to any method of determining the nucleotide composition of a nucleic acid of interest. Nucleic acid detection assay include but are not limited to, DNA sequencing methods, probe hybridization methods, structure specific cleavage assays (e.g., the INVADER assay, (Hologic, Inc.) and are described, e.g., in U.S. Pat. Nos. 5,846,717, 5,985,557, 5,994,069, 6,001,567, 6,090,543, and 6,872,816; Lyamichev et al., Nat. Biotech., 17:292 (1999), Hall et al., PNAS, USA, 97:8272 (2000), and U.S. Pat. No. 9,096,893, each of which is herein incorporated by reference in its entirety for all purposes); enzyme mismatch cleavage methods (e.g., Variagenics, U.S. Pat. Nos. 6,110,684, 5,958,692, 5,851,770, herein incorporated by reference in their entireties); polymerase chain reaction (PCR), described above; branched hybridization methods (e.g., Chiron, U.S. Pat. Nos. 5,849,481, 5,710,264, 5,124,246, and 5,624,802, herein incorporated by reference in their entireties); rolling circle replication (e.g., U.S. Pat. Nos. 6,210,884, 6,183,960 and 6,235,502, herein incorporated by reference in their entireties); NASBA (e.g., U.S. Pat. No. 5,409,818, herein incorporated by reference in its entirety); molecular beacon technology (e.g., U.S. Pat. No. 6,150,097, herein incorporated by reference in its entirety); E-sensor technology (Motorola, U.S. Pat. Nos. 6,248,229, 6,221,583, 6,013,170, and 6,063,573, herein incorporated by reference in their entireties); cycling probe technology (e.g., U.S. Pat. Nos. 5,403,711, 5,011,769, and 5,660,988, herein incorporated by reference in their entireties); Dade Behring signal amplification methods (e.g., U.S. Pat. Nos. 6,121,001, 6,110,677, 5,914,230, 5,882,867, and 5,792,614, herein incorporated by reference in their entireties); ligase chain reaction (e.g., Baranay Proc. Natl. Acad. Sci USA 88, 189-93 (1991)); and sandwich hybridization methods (e.g., U.S. Pat. No. 5,288,609, herein incorporated by reference in its entirety). 
     The term “amplifiable nucleic acid” refers to a nucleic acid that may be amplified by any amplification method. It is contemplated that “amplifiable nucleic acid” will usually comprise “sample template.” 
     The term “sample template” refers to nucleic acid originating from a sample that is analyzed for the presence of “target” (defined below). In contrast, “background template” is used in reference to nucleic acid other than sample template that may or may not be present in a sample. Background template is most often inadvertent. It may be the result of carryover or it may be due to the presence of nucleic acid contaminants sought to be purified away from the sample. For example, nucleic acids from organisms other than those to be detected may be present as background in a test sample. 
     The term “primer” refers to an oligonucleotide, whether occurring naturally as, e.g., a nucleic acid fragment from a restriction digest, or produced synthetically, that is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product that is complementary to a nucleic acid template strand is induced, (e.g., in the presence of nucleotides and an inducing agent such as a DNA polymerase, and at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer, and the use of the method. 
     The term “probe” refers to an oligonucleotide (e.g., a sequence of nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly, or by PCR amplification, that is capable of hybridizing to another oligonucleotide of interest. A probe may be single-stranded or double-stranded. Probes are useful in the detection, identification, and isolation of particular gene sequences (e.g., a “capture probe”). It is contemplated that any probe used in the present invention may, in some embodiments, be labeled with any “reporter molecule,” so that is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label. 
     The term “target,” as used herein refers to a nucleic acid sought to be sorted out from other nucleic acids, e.g., by probe binding, amplification, isolation, capture, etc. For example, when used in reference to the polymerase chain reaction, “target” refers to the region of nucleic acid bounded by the primers used for polymerase chain reaction, while when used in an assay in which target DNA is not amplified, e.g., in some embodiments of an invasive cleavage assay, a target comprises the site at which a probe and invasive oligonucleotides (e.g., INVADER oligonucleotide) bind to form an invasive cleavage structure, such that the presence of the target nucleic acid can be detected. A “segment” is defined as a region of nucleic acid within the target sequence. 
     Accordingly, as used herein, “non-target”, e.g., as it is used to describe a nucleic acid such as a DNA, refers to nucleic acid that may be present in a reaction, but that is not the subject of detection or characterization by the reaction. In some embodiments, non-target nucleic acid may refer to nucleic acid present in a sample that does not, e.g., contain a target sequence, while in some embodiments, non-target may refer to exogenous nucleic acid, i.e., nucleic acid that does not originate from a sample containing or suspected of containing a target nucleic acid, and that is added to a reaction, e.g., to normalize the activity of an enzyme (e.g., polymerase) to reduce variability in the performance of the enzyme in the reaction. As used herein, “methylation” refers to cytosine methylation at positions C5 or N4 of cytosine, the N6 position of adenine, or other types of nucleic acid methylation. In vitro amplified DNA is usually unmethylated because typical in vitro DNA amplification methods do not retain the methylation pattern of the amplification template. However, “unmethylated DNA” or “methylated DNA” can also refer to amplified DNA whose original template was unmethylated or methylated, respectively. 
     As used herein, the term “amplification reagents” refers to those reagents (deoxyribonucleoside triphosphates, buffer, etc.), needed for amplification except for primers, nucleic acid template, and the amplification enzyme. Typically, amplification reagents along with other reaction components are placed and contained in a reaction vessel. 
     As used herein, the term “control” when used in reference to nucleic acid detection or analysis refers to a nucleic acid having known features (e.g., known sequence, known copy-number per cell), for use in comparison to an experimental target (e.g., a nucleic acid of unknown concentration). A control may be an endogenous, preferably invariant gene against which a test or target nucleic acid in an assay can be normalized. Such normalizing controls for sample-to-sample variations that may occur in, for example, sample processing, assay efficiency, etc., and allows accurate sample-to-sample data comparison. Genes that find use for normalizing nucleic acid detection assays on human samples include, e.g., β-actin, ZDHHC1, and B3GALT6 (see, e.g., U.S. patent application Ser. Nos 14/966,617 and 62/364,082, each incorporated herein by reference. 
     Controls may also be external. For example, in quantitative assays such as qPCR, QuARTS, etc., a “calibrator” or “calibration control” is a nucleic acid of known sequence, e.g., having the same sequence as a portion of an experimental target nucleic acid, and a known concentration or series of concentrations (e.g., a serially diluted control target for generation of calibration curved in quantitative PCR). Typically, calibration controls are analyzed using the same reagents and reaction conditions as are used on an experimental DNA. In certain embodiments, the measurement of the calibrators is done at the same time, e.g., in the same thermal cycler, as the experimental assay. In preferred embodiments, multiple calibrators may be included in a single plasmid, such that the different calibrator sequences are easily provided in equimolar amounts. In particularly preferred embodiments, plasmid calibrators are digested, e.g., with one or more restriction enzymes, to release calibrator portion from the plasmid vector. See, e.g., WO 2015/066695, which is included herein by reference. 
     As used herein “ZDHHC1” refers to a gene encoding a protein characterized as a zinc finger, DHHC-type containing 1, located in human DNA on Chr 16 (16q22.1) and belonging to the DHHC palmitoyltransferase family. As used herein, “methylation” refers to cytosine methylation at positions C5 or N4 of cytosine, the N6 position of adenine, or other types of nucleic acid methylation. In vitro amplified DNA is usually unmethylated because typical in vitro DNA amplification methods do not retain the methylation pattern of the amplification template. However, “unmethylated DNA” or “methylated DNA” can also refer to amplified DNA whose original template was unmethylated or methylated, respectively. 
     Accordingly, as used herein a “methylated nucleotide” or a “methylated nucleotide base” refers to the presence of a methyl moiety on a nucleotide base, where the methyl moiety is not present in a recognized typical nucleotide base. For example, cytosine does not contain a methyl moiety on its pyrimidine ring, but 5-methylcytosine contains a methyl moiety at position 5 of its pyrimidine ring. Therefore, cytosine is not a methylated nucleotide and 5-methylcytosine is a methylated nucleotide. In another example, thymine contains a methyl moiety at position 5 of its pyrimidine ring; however, for purposes herein, thymine is not considered a methylated nucleotide when present in DNA since thymine is a typical nucleotide base of DNA. 
     As used herein, a “methylated nucleic acid molecule” refers to a nucleic acid molecule that contains one or more methylated nucleotides. 
     As used herein, a “methylation state”, “methylation profile”, and “methylation status” of a nucleic acid molecule refers to the presence of absence of one or more methylated nucleotide bases in the nucleic acid molecule. For example, a nucleic acid molecule containing a methylated cytosine is considered methylated (e.g., the methylation state of the nucleic acid molecule is methylated). A nucleic acid molecule that does not contain any methylated nucleotides is considered unmethylated. 
     In some embodiments, the sample is a stool sample, a tissue sample (e.g., skin tissue), a blood sample (e.g., plasma sample, whole blood sample, serum sample), or a urine sample. In some embodiments, the sample comprises blood, serum, plasma, gastric secretions, pancreatic juice, a cerebral spinal fluid (CSF) sample, a gastrointestinal biopsy sample, and/or cells recovered from stool. In some embodiments, the subject is human. The sample may include cells, secretions, or tissues from the lymph gland, breast, liver, bile ducts, pancreas, stomach, colon, rectum, esophagus, small intestine, appendix, duodenum, polyps, gall bladder, anus, and/or peritoneum. In some embodiments, the sample comprises cellular fluid, ascites, urine, feces, gastric section, pancreatic fluid, fluid obtained during endoscopy, blood. 
     As used herein, “methylation frequency” or “methylation percent (%)” refer to the number of instances in which a molecule or locus is methylated relative to the number of instances the molecule or locus is unmethylated. 
     As such, the methylation state describes the state of methylation of a nucleic acid (e.g., a genomic sequence). In addition, the methylation state refers to the characteristics of a nucleic acid segment at a particular genomic locus relevant to methylation. Such characteristics include, but are not limited to, whether any of the cytosine (C) residues within this DNA sequence are methylated, the location of methylated C residue(s), the frequency or percentage of methylated C throughout any particular region of a nucleic acid, and allelic differences in methylation due to, e.g., difference in the origin of the alleles. The terms “methylation state”, “methylation profile”, and “methylation status” also refer to the relative concentration, absolute concentration, or pattern of methylated C or unmethylated C throughout any particular region of a nucleic acid in a biological sample. For example, if the cytosine (C) residue(s) within a nucleic acid sequence are methylated it may be referred to as “hypermethylated” or having “increased methylation”, whereas if the cytosine (C) residue(s) within a DNA sequence are not methylated it may be referred to as “hypomethylated” or having “decreased methylation”. Likewise, if the cytosine (C) residue(s) within a nucleic acid sequence are methylated as compared to another nucleic acid sequence (e.g., from a different region or from a different individual, etc.) that sequence is considered hypermethylated or having increased methylation compared to the other nucleic acid sequence. Alternatively, if the cytosine (C) residue(s) within a DNA sequence are not methylated as compared to another nucleic acid sequence (e.g., from a different region or from a different individual, etc.) that sequence is considered hypomethylated or having decreased methylation compared to the other nucleic acid sequence. Additionally, the term “methylation pattern” as used herein refers to the collective sites of methylated and unmethylated nucleotides over a region of a nucleic acid. Two nucleic acids may have the same or similar methylation frequency or methylation percent but have different methylation patterns when the number of methylated and unmethylated nucleotides are the same or similar throughout the region but the locations of methylated and unmethylated nucleotides are different. Sequences are said to be “differentially methylated” or as having a “difference in methylation” or having a “different methylation state” when they differ in the extent (e.g., one has increased or decreased methylation relative to the other), frequency, or pattern of methylation. The term “differential methylation” refers to a difference in the level or pattern of nucleic acid methylation in a cancer positive sample as compared with the level or pattern of nucleic acid methylation in a cancer negative sample. It may also refer to the difference in levels or patterns between patients that have recurrence of cancer after surgery versus patients who not have recurrence. Differential methylation and specific levels or patterns of DNA methylation are prognostic and predictive biomarkers, e.g., once the correct cut-off or predictive characteristics have been defined. 
     Methylation state frequency can be used to describe a population of individuals or a sample from a single individual. For example, a nucleotide locus having a methylation state frequency of 50% is methylated in 50% of instances and unmethylated in 50% of instances. Such a frequency can be used, for example, to describe the degree to which a nucleotide locus or nucleic acid region is methylated in a population of individuals or a collection of nucleic acids. Thus, when methylation in a first population or pool of nucleic acid molecules is different from methylation in a second population or pool of nucleic acid molecules, the methylation state frequency of the first population or pool will be different from the methylation state frequency of the second population or pool. Such a frequency also can be used, for example, to describe the degree to which a nucleotide locus or nucleic acid region is methylated in a single individual. For example, such a frequency can be used to describe the degree to which a group of cells from a tissue sample are methylated or unmethylated at a nucleotide locus or nucleic acid region. 
     As used herein a “nucleotide locus” refers to the location of a nucleotide in a nucleic acid molecule. A nucleotide locus of a methylated nucleotide refers to the location of a methylated nucleotide in a nucleic acid molecule. 
     Typically, methylation of human DNA occurs on a dinucleotide sequence including an adjacent guanine and cytosine where the cytosine is located 5′ of the guanine (also termed CpG dinucleotide sequences). Most cytosines within the CpG dinucleotides are methylated in the human genome, however some remain unmethylated in specific CpG dinucleotide rich genomic regions, known as CpG islands (see, e.g, Antequera et al. (1990)  Cell  62: 503-514). 
     As used herein, a “CpG island” refers to a G:C-rich region of genomic DNA containing an increased number of CpG dinucleotides relative to total genomic DNA. A CpG island can be at least 100, 200, or more base pairs in length, where the G:C content of the region is at least 50% and the ratio of observed CpG frequency over expected frequency is 0.6; in some instances, a CpG island can be at least 500 base pairs in length, where the G:C content of the region is at least 55%) and the ratio of observed CpG frequency over expected frequency is 0.65. The observed CpG frequency over expected frequency can be calculated according to the method provided in Gardiner-Garden et al (1987)  J Mol. Biol.  196: 261-281. For example, the observed CpG frequency over expected frequency can be calculated according to the formula R=(A×B)/(C×D), where R is the ratio of observed CpG frequency over expected frequency, A is the number of CpG dinucleotides in an analyzed sequence, B is the total number of nucleotides in the analyzed sequence, C is the total number of C nucleotides in the analyzed sequence, and D is the total number of G nucleotides in the analyzed sequence. Methylation state is typically determined in CpG islands, e.g., at promoter regions. It will be appreciated though that other sequences in the human genome are prone to DNA methylation such as CpA and CpT (see Ramsahoye (2000) Proc. Natl. Acad. Sci. USA 97: 5237-5242; Salmon and Kaye (1970) Biochim. Biophys. Acta. 204: 340-351; Grafstrom (1985) Nucleic Acids Res. 13: 2827-2842; Nyce (1986) Nucleic Acids Res. 14: 4353-4367; Woodcock (1987) Biochem. Biophys. Res. Commun. 145: 888-894). 
     As used herein, a “methylation-specific reagent” refers to a reagent that modifies a nucleotide of the nucleic acid molecule as a function of the methylation state of the nucleic acid molecule, or a methylation-specific reagent, refers to a compound or composition or other agent that can change the nucleotide sequence of a nucleic acid molecule in a manner that reflects the methylation state of the nucleic acid molecule. Methods of treating a nucleic acid molecule with such a reagent can include contacting the nucleic acid molecule with the reagent, coupled with additional steps, if desired, to accomplish the desired change of nucleotide sequence. Such methods can be applied in a manner in which unmethylated nucleotides (e.g., each unmethylated cytosine) is modified to a different nucleotide. For example, in some embodiments, such a reagent can deaminate unmethylated cytosine nucleotides to produce deoxy uracil residues. Examples of such reagents include, but are not limited to, a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and a bisulfite reagent. 
     A change in the nucleic acid nucleotide sequence by a methylation-specific reagent can also result in a nucleic acid molecule in which each methylated nucleotide is modified to a different nucleotide. 
     As used herein, the term “UDP glucose modified with a chemoselective group” refers to a uridine diphosphoglucose molecule that has been functionalized, particularly at the 6-hydroxyl position, with a functional group capable of reaction with an affinity tag via click chemistry. 
     The term “oxidized 5-methylcytosine” refers to an oxidized 5-methylcytosine residue that has been oxidized at the 5-position. Oxidized 5-methylcytosine residues thus include 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxymethylcytosine. The oxidized 5-methylcytosine residues that undergo reaction with an organic borane according to one embodiment of the invention are 5-formylcytosine and 5-carboxymethylcytosine. 
     The term “methylation assay” refers to any assay for determining the methylation state of one or more CpG dinucleotide sequences within a sequence of a nucleic acid. 
     The term “MS AP-PCR” (Methylation-Sensitive Arbitrarily-Primed Polymerase Chain Reaction) refers to the art-recognized technology that allows for a global scan of the genome using CG-rich primers to focus on the regions most likely to contain CpG dinucleotides, and described by Gonzalgo et al. (1997)  Cancer Research  57: 594-599. 
     The term “MethyLight™” refers to the art-recognized fluorescence-based real-time PCR technique described by Eads et al. (1999)  Cancer Res.  59: 2302-2306. 
     The term “HeavyMethyl™” refers to an assay wherein methylation specific blocking probes (also referred to herein as blockers) covering CpG positions between, or covered by, the amplification primers enable methylation-specific selective amplification of a nucleic acid sample. 
     The term “HeavyMethyl™ MethyLight™” assay refers to a HeavyMethyl™ MethyLight™ assay, which is a variation of the MethyLight™ assay, wherein the MethyLight™ assay is combined with methylation specific blocking probes covering CpG positions between the amplification primers. 
     The term “Ms-SNuPE” (Methylation-sensitive Single Nucleotide Primer Extension) refers to the art-recognized assay described by Gonzalgo &amp; Jones (1997)  Nucleic Acids Res.  25: 2529-2531. 
     The term “MSP” (Methylation-specific PCR) refers to the art-recognized methylation assay described by Herman et al. (1996)  Proc. Nat. Acad. Sci. USA  93: 9821-9826, and by U.S. Pat. No. 5,786,146. 
     The term “COBRA” (Combined Bisulfite Restriction Analysis) refers to the art-recognized methylation assay described by Xiong &amp; Laird (1997)  Nucleic Acids Res.  25: 2532-2534. 
     The term “MCA” (Methylated CpG Island Amplification) refers to the methylation assay described by Toyota et al. (1999)  Cancer Res.  59: 2307-12, and in WO 00/26401A1. 
     As used herein, a “selected nucleotide” refers to one nucleotide of the four typically occurring nucleotides in a nucleic acid molecule (C, G, T, and A for DNA and C, G, U, and A for RNA), and can include methylated derivatives of the typically occurring nucleotides (e.g., when C is the selected nucleotide, both methylated and unmethylated C are included within the meaning of a selected nucleotide), whereas a methylated selected nucleotide refers specifically to a methylated typically occurring nucleotide and an unmethylated selected nucleotides refers specifically to an unmethylated typically occurring nucleotide. 
     The term “methylation-specific restriction enzyme” refers to a restriction enzyme that selectively digests a nucleic acid dependent on the methylation state of its recognition site. In the case of a restriction enzyme that specifically cuts if the recognition site is not methylated or is hemi-methylated (a methylation-sensitive enzyme), the cut will not take place (or will take place with a significantly reduced efficiency) if the recognition site is methylated on one or both strands. In the case of a restriction enzyme that specifically cuts only if the recognition site is methylated (a methylation-dependent enzyme), the cut will not take place (or will take place with a significantly reduced efficiency) if the recognition site is not methylated. Preferred are methylation-specific restriction enzymes, the recognition sequence of which contains a CG dinucleotide (for instance a recognition sequence such as CGCG or CCCGGG). Further preferred for some embodiments are restriction enzymes that do not cut if the cytosine in this dinucleotide is methylated at the carbon atom C5. 
     As used herein, a “different nucleotide” refers to a nucleotide that is chemically different from a selected nucleotide, typically such that the different nucleotide has Watson-Crick base-pairing properties that differ from the selected nucleotide, whereby the typically occurring nucleotide that is complementary to the selected nucleotide is not the same as the typically occurring nucleotide that is complementary to the different nucleotide. For example, when C is the selected nucleotide, U or T can be the different nucleotide, which is exemplified by the complementarity of C to G and the complementarity of U or T to A. As used herein, a nucleotide that is complementary to the selected nucleotide or that is complementary to the different nucleotide refers to a nucleotide that base-pairs, under high stringency conditions, with the selected nucleotide or different nucleotide with higher affinity than the complementary nucleotide&#39;s base-paring with three of the four typically occurring nucleotides. An example of complementarity is Watson-Crick base pairing in DNA (e.g., A-T and C-G) and RNA (e.g., A-U and C-G). Thus, for example, G base-pairs, under high stringency conditions, with higher affinity to C than G base-pairs to G, A, or T and, therefore, when C is the selected nucleotide, G is a nucleotide complementary to the selected nucleotide. 
     As used herein, the “sensitivity” of a given marker (or set of markers used together) refers to the percentage of samples that report a DNA methylation value above a threshold value that distinguishes between neoplastic and non-neoplastic samples. In some embodiments, a positive is defined as a histology-confirmed neoplasia that reports a DNA methylation value above a threshold value (e.g., the range associated with disease), and a false negative is defined as a histology-confirmed neoplasia that reports a DNA methylation value below the threshold value (e.g., the range associated with no disease). The value of sensitivity, therefore, reflects the probability that a DNA methylation measurement for a given marker obtained from a known diseased sample will be in the range of disease-associated measurements. As defined here, the clinical relevance of the calculated sensitivity value represents an estimation of the probability that a given marker would detect the presence of a clinical condition when applied to a subject with that condition. 
     As used herein, the “specificity” of a given marker (or set of markers used together) refers to the percentage of non-neoplastic samples that report a DNA methylation value below a threshold value that distinguishes between neoplastic and non-neoplastic samples. In some embodiments, a negative is defined as a histology-confirmed non-neoplastic sample that reports a DNA methylation value below the threshold value (e.g., the range associated with no disease) and a false positive is defined as a histology-confirmed non-neoplastic sample that reports a DNA methylation value above the threshold value (e.g., the range associated with disease). The value of specificity, therefore, reflects the probability that a DNA methylation measurement for a given marker obtained from a known non-neoplastic sample will be in the range of non-disease associated measurements. As defined here, the clinical relevance of the calculated specificity value represents an estimation of the probability that a given marker would detect the absence of a clinical condition when applied to a patient without that condition. 
     The term “AUC” as used herein is an abbreviation for the “area under a curve”. In particular it refers to the area under a Receiver Operating Characteristic (ROC) curve. The ROC curve is a plot of the true positive rate against the false positive rate for the different possible cut points of a diagnostic test. It shows the trade-off between sensitivity and specificity depending on the selected cut point (any increase in sensitivity will be accompanied by a decrease in specificity). The area under an ROC curve (AUC) is a measure for the accuracy of a diagnostic test (the larger the area the better; the optimum is 1; a random test would have a ROC curve lying on the diagonal with an area of 0.5; for reference: J. P. Egan. (1975)  Signal Detection Theory and ROC Analysis , Academic Press, New York). 
     The term “neoplasm” as used herein refers to any new and abnormal growth of tissue. Thus, a neoplasm can be a premalignant neoplasm or a malignant neoplasm. 
     The term “neoplasm-specific marker,” as used herein, refers to any biological material or element that can be used to indicate the presence of a neoplasm. Examples of biological materials include, without limitation, nucleic acids, polypeptides, carbohydrates, fatty acids, cellular components (e.g., cell membranes and mitochondria), and whole cells. In some instances, markers are particular nucleic acid regions (e.g., genes, intragenic regions, specific loci, etc.). Regions of nucleic acid that are markers may be referred to, e.g., as “marker genes,” “marker regions,” “marker sequences,” “marker loci,” etc. 
     As used herein, the term “adenoma” refers to a benign tumor of glandular origin. Although these growths are benign, over time they may progress to become malignant. 
     The term “pre-cancerous” or “pre-neoplastic” and equivalents thereof refer to any cellular proliferative disorder that is undergoing malignant transformation. 
     A “site” of a neoplasm, adenoma, cancer, etc. is the tissue, organ, cell type, anatomical area, body part, etc. in a subject&#39;s body where the neoplasm, adenoma, cancer, etc. is located. 
     As used herein, a “diagnostic” test application includes the detection or identification of a disease state or condition of a subject, determining the likelihood that a subject will contract a given disease or condition, determining the likelihood that a subject with a disease or condition will respond to therapy, determining the prognosis of a subject with a disease or condition (or its likely progression or regression), and determining the effect of a treatment on a subject with a disease or condition. For example, a diagnostic can be used for detecting the presence or likelihood of a subject contracting a neoplasm or the likelihood that such a subject will respond favorably to a compound (e.g., a pharmaceutical, e.g., a drug) or other treatment. 
     The term “isolated” when used in relation to a nucleic acid, as in “an isolated oligonucleotide” refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acid is present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids, such as DNA and RNA, are found in the state they exist in nature. Examples of non-isolated nucleic acids include: a given DNA sequence (e.g., a gene) found on the host cell chromosome in proximity to neighboring genes; RNA sequences, such as a specific mRNA sequence encoding a specific protein, found in the cell as a mixture with numerous other mRNAs which encode a multitude of proteins. However, isolated nucleic acid encoding a particular protein includes, by way of example, such nucleic acid in cells ordinarily expressing the protein, where the nucleic acid is in a chromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature. The isolated nucleic acid or oligonucleotide may be present in single-stranded or double-stranded form. When an isolated nucleic acid or oligonucleotide is to be utilized to express a protein, the oligonucleotide will contain at a minimum the sense or coding strand (i.e., the oligonucleotide may be single-stranded), but may contain both the sense and anti-sense strands (i.e., the oligonucleotide may be double-stranded). An isolated nucleic acid may, after isolation from its natural or typical environment, be combined with other nucleic acids or molecules. For example, an isolated nucleic acid may be present in a host cell into which it has been placed, e.g., for heterologous expression. 
     The term “purified” refers to molecules, either nucleic acid or amino acid sequences that are removed from their natural environment, isolated, or separated. An “isolated nucleic acid sequence” may therefore be a purified nucleic acid sequence. “Substantially purified” molecules are at least 60% free, preferably at least 75% free, and more preferably at least 90% free from other components with which they are naturally associated. As used herein, the terms “purified” or “to purify” also refer to the removal of contaminants from a sample. The removal of contaminating proteins results in an increase in the percent of polypeptide or nucleic acid of interest in the sample. In another example, recombinant polypeptides are expressed in plant, bacterial, yeast, or mammalian host cells and the polypeptides are purified by the removal of host cell proteins; the percent of recombinant polypeptides is thereby increased in the sample. 
     The term “composition comprising” a given polynucleotide sequence or polypeptide refers broadly to any composition containing the given polynucleotide sequence or polypeptide. The composition may comprise an aqueous solution containing salts (e.g., NaCl), detergents (e.g., SDS), and other components (e.g., Denhardt&#39;s solution, dry milk, salmon sperm DNA, etc.). 
     The term “sample” is used in its broadest sense. In one sense it can refer to an animal cell or tissue. In another sense, it refers to a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from plants or animals (including humans) and encompass fluids, solids, tissues, and gases. Environmental samples include environmental material such as surface matter, soil, water, and industrial samples. These examples are not to be construed as limiting the sample types applicable to the present invention. 
     As used herein, a “remote sample” as used in some contexts relates to a sample collected from a site that is not the cell, tissue, or organ source of the sample. 
     As used herein, the terms “patient” or “subject” refer to organisms to be subject to various tests provided by the technology. The term “subject” includes animals, preferably mammals, including humans. In a preferred embodiment, the subject is a primate. In an even more preferred embodiment, the subject is a human. Further with respect to diagnostic methods, a preferred subject is a vertebrate subject. A preferred vertebrate is warm-blooded; a preferred warm-blooded vertebrate is a mammal. A preferred mammal is most preferably a human. As used herein, the term “subject’ includes both human and animal subjects. Thus, veterinary therapeutic uses are provided herein. As such, the present technology provides for the diagnosis of mammals such as humans, as well as those mammals of importance due to being endangered, such as Siberian tigers; of economic importance, such as animals raised on farms for consumption by humans; and/or animals of social importance to humans, such as animals kept as pets or in zoos. Examples of such animals include but are not limited to: carnivores such as cats and dogs; swine, including pigs, hogs, and wild boars; ruminants and/or ungulates such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels; pinnipeds; and horses. Thus, also provided is the diagnosis and treatment of livestock, including, but not limited to, domesticated swine, ruminants, ungulates, horses (including race horses), and the like. The presently-disclosed subject matter further includes a system for diagnosing a lung cancer in a subject. The system can be provided, for example, as a commercial kit that can be used to screen for a risk of lung cancer or diagnose a lung cancer in a subject from whom a biological sample has been collected. An exemplary system provided in accordance with the present technology includes assessing the methylation state of a marker described herein. 
     As used herein, the term “kit” refers to any delivery system for delivering materials. In the context of reaction assays, such delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. in the appropriate containers) and/or supporting materials (e.g., buffers, written instructions for performing the assay etc.) from one location to another. For example, kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and/or supporting materials. As used herein, the term “fragmented kit” refers to delivery systems comprising two or more separate containers that each contain a subportion of the total kit components. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain an enzyme for use in an assay, while a second container contains oligonucleotides. The term “fragmented kit” is intended to encompass kits containing Analyte specific reagents (ASR&#39;s) regulated under section 520(e) of the Federal Food, Drug, and Cosmetic Act, but are not limited thereto. Indeed, any delivery system comprising two or more separate containers that each contains a subportion of the total kit components are included in the term “fragmented kit.” In contrast, a “combined kit” refers to a delivery system containing all of the components of a reaction assay in a single container (e.g., in a single box housing each of the desired components). The term “kit” includes both fragmented and combined kits. 
     As used herein, the term “nevi” or “mole” refers to a typically noncancerous skin growth made up of cells (melanocytes or nevus cells) that produce color (pigment). Moles can appear anywhere on the skin, alone or in groups. 
     As used herein, the term “lesion” refers to a mole that is under examination (e.g., is suspected of being cancerous or has been diagnosed as cancerous) and may or may not be cancerous. In some embodiments, “lesion” is used interchangeably with “mole” or “nevi.” 
     As used herein, the term “melanoma” or “malignant melanoma” refers to a serious form of skin cancer that may affect the skin only or may spread (metastasize) through the blood or lymph systems to organs and bones. Melanoma can develop in an existing mole or other mark on the skin or on unmarked skin. 
     As used herein, the term “metastatic melanoma” refers to melanoma that has spread to other tissues or organs. 
     As used herein, the term “information” refers to any collection of facts or data. In reference to information stored or processed using a computer system(s), including but not limited to internets, the term refers to any data stored in any format (e.g., analog, digital, optical, etc.). As used herein, the term “information related to a subject” refers to facts or data pertaining to a subject (e.g., a human, plant, or animal). The term “genomic information” refers to information pertaining to a genome including, but not limited to, nucleic acid sequences, genes, percentage methylation, allele frequencies, RNA expression levels, protein expression, phenotypes correlating to genotypes, etc. “Allele frequency information” refers to facts or data pertaining to allele frequencies, including, but not limited to, allele identities, statistical correlations between the presence of an allele and a characteristic of a subject (e.g., a human subject), the presence or absence of an allele in an individual or population, the percentage likelihood of an allele being present in an individual having one or more particular characteristics, etc. 
     DETAILED DESCRIPTION 
     In this detailed description of the various embodiments, for purposes of explanation, numerous specific details are set forth to provide a thorough understanding of the embodiments disclosed. One skilled in the art will appreciate, however, that these various embodiments may be practiced with or without these specific details. In other instances, structures and devices are shown in block diagram form. Furthermore, one skilled in the art can readily appreciate that the specific sequences in which methods are presented and performed are illustrative and it is contemplated that the sequences can be varied and still remain within the spirit and scope of the various embodiments disclosed herein. 
     Provided herein is technology for melanoma screening and particularly, but not exclusively, to methods, compositions, and related uses for detecting the presence of melanoma and/or specific forms of melanoma (e.g., metastatic melanoma, primary cutaneous melanoma). As the technology is described herein, the section headings used are for organizational purposes only and are not to be construed as limiting the subject matter in any way. 
     Indeed, as described in Examples I and II, experiments conducted during the course for identifying embodiments for the present invention identified a novel set of 331 differentially methylated regions (DMRs) for discriminating melanoma derived DNA from non-neoplastic control DNA. From these 331 novel DNA methylation markers, further experiments identified markers capable of distinguishing different types of melanoma from normal tissue. For example, separate sets of DMRs were identified capable of distinguishing 1) metastatic melanoma tissue from normal tissue, and 2) primary cutaneous melanoma tissue from normal tissue. 
     Although the disclosure herein refers to certain illustrated embodiments, it is to be understood that these embodiments are presented by way of example and not by way of limitation. 
     In particular aspects, the present technology provides compositions and methods for identifying, determining, and/or classifying a cancer such as melanoma and/or a sub-type of melanoma (e.g., metastatic melanoma, primary cutaneous melanoma). The methods comprise determining the methylation status of at least one methylation marker in a biological sample isolated from a subject (e.g., a stool sample; skin tissue sample; fine needle aspirate, deep tissue sample, plasma sample, serum sample, whole blood sample), wherein a change in the methylation state of the marker is indicative of the presence, class, or site of melanoma and/or a sub-type thereof. Particular embodiments relate to markers comprising a differentially methylated region (DMR, e.g., DMR 1-331, see Tables 1A, 2A, 7A, 8A, and 9) that are used for diagnosis (e.g., screening) of melanoma and various types of melanoma (e.g., metastatic melanoma, primary cutaneous melanoma). 
     In addition to embodiments wherein the methylation analysis of at least one marker, a region of a marker, or a base of a marker comprising a DMR (e.g., DMR 1-331) provided herein and listed in Tables 1A, 2A, 7A, 8A, and 9 is analyzed, the technology also provides panels of markers comprising at least one marker, region of a marker, or base of a marker comprising a DMR with utility for the detection of cancers, in particular melanoma. 
     Some embodiments of the technology are based upon the analysis of the CpG methylation status of at least one marker, region of a marker, or base of a marker comprising a DMR. 
     In some embodiments, the present technology provides for the use of a reagent that modifies DNA in a methylation-specific manner (e.g., a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and a bisulfite reagent) in combination with one or more methylation assays to determine the methylation status of CpG dinucleotide sequences within at least one marker comprising a DMR (e.g., DMR 1-331, see Tables 1A, 2A, 7A, 8A, and 9). Genomic CpG dinucleotides can be methylated or unmethylated (alternatively known as up- and down-methylated respectively). However, the methods of the present invention are suitable for the analysis of biological samples of a heterogeneous nature, e.g., a low concentration of tumor cells, or biological materials therefrom, within a background of a remote sample (e.g., blood, organ effluent, or stool). Accordingly, when analyzing the methylation status of a CpG position within such a sample one may use a quantitative assay for determining the level (e.g., percent, fraction, ratio, proportion, or degree) of methylation at a particular CpG position. 
     According to the present technology, determination of the methylation status of CpG dinucleotide sequences in markers comprising a DMR has utility both in the diagnosis and characterization of cancers such as melanoma. 
     Combinations of Markers 
     A frequently used method for analyzing a nucleic acid for the presence of 5-methylcytosine is based upon the bisulfite method described by Frommer, et al. for the detection of 5-methylcytosines in DNA (Frommer et al. (1992)  Proc. Natl. Acad. Sci. USA  89: 1827-31 explicitly incorporated herein by reference in its entirety for all purposes) or variations thereof. The bisulfite method of mapping 5-methylcytosines is based on the observation that cytosine, but not 5-methylcytosine, reacts with hydrogen sulfite ion (also known as bisulfite). The reaction is usually performed according to the following steps: first, cytosine reacts with hydrogen sulfite to form a sulfonated cytosine. Next, spontaneous deamination of the sulfonated reaction intermediate results in a sulfonated uracil. Finally, the sulfonated uracil is desulfonated under alkaline conditions to form uracil. Detection is possible because uracil base pairs with adenine (thus behaving like thymine), whereas 5-methylcytosine base pairs with guanine (thus behaving like cytosine). This makes the discrimination of methylated cytosines from non-methylated cytosines possible by, e.g., bisulfite genomic sequencing (Grigg G, &amp; Clark S, Bioessays (1994) 16: 431-36; Grigg G, DNA Seq. (1996) 6: 189-98), methylation-specific PCR (MSP) as is disclosed, e.g., in U.S. Pat. No. 5,786,146, or using an assay comprising sequence-specific probe cleavage, e.g., a QuARTS flap endonuclease assay (see, e.g., Zou et al. (2010) “Sensitive quantification of methylated markers with a novel methylation specific technology”  Clin Chem  56: A199; and in U.S. Pat. Nos. 8,361,720; 8,715,937; 8,916,344; and 9,212,392). 
     Some conventional technologies are related to methods comprising enclosing the DNA to be analyzed in an agarose matrix, thereby preventing the diffusion and renaturation of the DNA (bisulfite only reacts with single-stranded DNA), and replacing precipitation and purification steps with a fast dialysis (Olek A, et al. (1996) “A modified and improved method for bisulfite based cytosine methylation analysis”  Nucleic Acids Res.  24: 5064-6). It is thus possible to analyze individual cells for methylation status, illustrating the utility and sensitivity of the method. An overview of conventional methods for detecting 5-methylcytosine is provided by Rein, T., et al. (1998)  Nucleic Acids Res.  26: 2255. 
     The bisulfite technique typically involves amplifying short, specific fragments of a known nucleic acid subsequent to a bisulfite treatment, then either assaying the product by sequencing (Olek &amp; Walter (1997)  Nat. Genet.  17: 275-6) or a primer extension reaction (Gonzalgo &amp; Jones (1997)  Nucleic Acids Res.  25: 2529-31; WO 95/00669; U.S. Pat. No. 6,251,594) to analyze individual cytosine positions. Some methods use enzymatic digestion (Xiong &amp; Laird (1997)  Nucleic Acids Res.  25: 2532-4). Detection by hybridization has also been described in the art (Olek et al., WO 99/28498). Additionally, use of the bisulfite technique for methylation detection with respect to individual genes has been described (Grigg &amp; Clark (1994)  Bioessays  16: 431-6; Zeschnigk et al. (1997)  Hum Mol Genet.  6: 387-95; Feil et al. (1994)  Nucleic Acids Res.  22: 695; Martin et al. (1995)  Gene  157: 261-4; WO 9746705; WO 9515373). 
     Various methylation assay procedures can be used in conjunction with bisulfite treatment according to the present technology. These assays allow for determination of the methylation state of one or a plurality of CpG dinucleotides (e.g., CpG islands) within a nucleic acid sequence. Such assays involve, among other techniques, sequencing of bisulfite-treated nucleic acid, PCR (for sequence-specific amplification), Southern blot analysis, and use of methylation-specific restriction enzymes, e.g., methylation-sensitive or methylation-dependent enzymes. 
     For example, genomic sequencing has been simplified for analysis of methylation patterns and 5-methylcytosine distributions by using bisulfite treatment (Frommer et al. (1992)  Proc. Natl. Acad. Sci. USA  89: 1827-1831). Additionally, restriction enzyme digestion of PCR products amplified from bisulfite-converted DNA finds use in assessing methylation state, e.g., as described by Sadri &amp; Hornsby (1997)  Nucl. Acids Res.  24: 5058-5059 or as embodied in the method known as COBRA (Combined Bisulfite Restriction Analysis) (Xiong &amp; Laird (1997)  Nucleic Acids Res.  25: 2532-2534). 
     COBRA™ analysis is a quantitative methylation assay useful for determining DNA methylation levels at specific loci in small amounts of genomic DNA (Xiong &amp; Laird, Nucleic Acids Res. 25:2532-2534, 1997). Briefly, restriction enzyme digestion is used to reveal methylation-dependent sequence differences in PCR products of sodium bisulfite-treated DNA. Methylation-dependent sequence differences are first introduced into the genomic DNA by standard bisulfite treatment according to the procedure described by Frommer et al. (Proc. Natl. Acad. Sci. USA 89:1827-1831, 1992). PCR amplification of the bisulfite converted DNA is then performed using primers specific for the CpG islands of interest, followed by restriction endonuclease digestion, gel electrophoresis, and detection using specific, labeled hybridization probes. Methylation levels in the original DNA sample are represented by the relative amounts of digested and undigested PCR product in a linearly quantitative fashion across a wide spectrum of DNA methylation levels. In addition, this technique can be reliably applied to DNA obtained from microdissected paraffin-embedded tissue samples. 
     Typical reagents (e.g., as might be found in a typical COBRA™-based kit) for COBRA™ analysis may include, but are not limited to: PCR primers for specific loci (e.g., specific genes, markers, DMR, regions of genes, regions of markers, bisulfite treated DNA sequence, CpG island, etc.); restriction enzyme and appropriate buffer; gene-hybridization oligonucleotide; control hybridization oligonucleotide; kinase labeling kit for oligonucleotide probe; and labeled nucleotides. Additionally, bisulfite conversion reagents may include: DNA denaturation buffer; sulfonation buffer; DNA recovery reagents or kits (e.g., precipitation, ultrafiltration, affinity column); desulfonation buffer; and DNA recovery components. Assays such as “MethyLight™” (a fluorescence-based real-time PCR technique) (Eads et al., Cancer Res. 59:2302-2306, 1999), Ms-SNuPE™ (Methylation-sensitive Single Nucleotide Primer Extension) reactions (Gonzalgo &amp; Jones, Nucleic Acids Res. 25:2529-2531, 1997), methylation-specific PCR (“MSP”; Herman et al., Proc. Natl. Acad. Sci. USA 93:9821-9826, 1996; U.S. Pat. No. 5,786,146), and methylated CpG island amplification (“MCA”; Toyota et al., Cancer Res. 59:2307-12, 1999) are used alone or in combination with one or more of these methods. 
     The “HeavyMethyl™” assay, technique is a quantitative method for assessing methylation differences based on methylation-specific amplification of bisulfite-treated DNA. Methylation-specific blocking probes (“blockers”) covering CpG positions between, or covered by, the amplification primers enable methylation-specific selective amplification of a nucleic acid sample. 
     The term “HeavyMethyl™ MethyLight™” assay refers to a HeavyMethyl™ MethyLight™ assay, which is a variation of the MethyLight™ assay, wherein the MethyLight™ assay is combined with methylation specific blocking probes covering CpG positions between the amplification primers. The HeavyMethyl™ assay may also be used in combination with methylation specific amplification primers. 
     Typical reagents (e.g., as might be found in a typical MethyLight™-based kit) for HeavyMethyl™ analysis may include, but are not limited to: PCR primers for specific loci (e.g., specific genes, markers, regions of genes, regions of markers, bisulfite treated DNA sequence, CpG island, or bisulfite treated DNA sequence or CpG island, etc.); blocking oligonucleotides; optimized PCR buffers and deoxynucleotides; and Taq polymerase. MSP (methylation-specific PCR) allows for assessing the methylation status of virtually any group of CpG sites within a CpG island, independent of the use of methylation-sensitive restriction enzymes (Herman et al. Proc. Natl. Acad. Sci. USA 93:9821-9826, 1996; U.S. Pat. No. 5,786,146). Briefly, DNA is modified by sodium bisulfite, which converts unmethylated, but not methylated cytosines, to uracil, and the products are subsequently amplified with primers specific for methylated versus unmethylated DNA. MSP requires only small quantities of DNA, is sensitive to 0.1% methylated alleles of a given CpG island locus, and can be performed on DNA extracted from paraffin-embedded samples. Typical reagents (e.g., as might be found in a typical MSP-based kit) for MSP analysis may include, but are not limited to: methylated and unmethylated PCR primers for specific loci (e.g., specific genes, markers, regions of genes, regions of markers, bisulfite treated DNA sequence, CpG island, etc.); optimized PCR buffers and deoxynucleotides, and specific probes. 
     The MethyLight™ assay is a high-throughput quantitative methylation assay that utilizes fluorescence-based real-time PCR (e.g., TaqMan®) that requires no further manipulations after the PCR step (Eads et al., Cancer Res. 59:2302-2306, 1999). Briefly, the MethyLight™ process begins with a mixed sample of genomic DNA that is converted, in a sodium bisulfite reaction, to a mixed pool of methylation-dependent sequence differences according to standard procedures (the bisulfite process converts unmethylated cytosine residues to uracil). Fluorescence-based PCR is then performed in a “biased” reaction, e.g., with PCR primers that overlap known CpG dinucleotides. Sequence discrimination occurs both at the level of the amplification process and at the level of the fluorescence detection process. 
     The MethyLight™ assay is used as a quantitative test for methylation patterns in a nucleic acid, e.g., a genomic DNA sample, wherein sequence discrimination occurs at the level of probe hybridization. In a quantitative version, the PCR reaction provides for a methylation specific amplification in the presence of a fluorescent probe that overlaps a particular putative methylation site. An unbiased control for the amount of input DNA is provided by a reaction in which neither the primers, nor the probe, overlie any CpG dinucleotides. Alternatively, a qualitative test for genomic methylation is achieved by probing the biased PCR pool with either control oligonucleotides that do not cover known methylation sites (e.g., a fluorescence-based version of the HeavyMethyl™ and MSP techniques) or with oligonucleotides covering potential methylation sites. 
     The MethyLight™ process is used with any suitable probe (e.g. a “TaqMan®” probe, a Lightcycler® probe, etc.) For example, in some applications double-stranded genomic DNA is treated with sodium bisulfite and subjected to one of two sets of PCR reactions using TaqMan® probes, e.g., with MSP primers and/or HeavyMethyl blocker oligonucleotides and a TaqMan® probe. The TaqMan® probe is dual-labeled with fluorescent “reporter” and “quencher” molecules and is designed to be specific for a relatively high GC content region so that it melts at about a 10° C. higher temperature in the PCR cycle than the forward or reverse primers. This allows the TaqMan® probe to remain fully hybridized during the PCR annealing/extension step. As the Taq polymerase enzymatically synthesizes a new strand during PCR, it will eventually reach the annealed TaqMan® probe. The Taq polymerase 5′ to 3′ endonuclease activity will then displace the TaqMan® probe by digesting it to release the fluorescent reporter molecule for quantitative detection of its now unquenched signal using a real-time fluorescent detection system. 
     Typical reagents (e.g., as might be found in a typical MethyLight™-based kit) for MethyLight™ analysis may include, but are not limited to: PCR primers for specific loci (e.g., specific genes, markers, regions of genes, regions of markers, bisulfite treated DNA sequence, CpG island, etc.); TaqMan® or Lightcycler® probes; optimized PCR buffers and deoxynucleotides; and Taq polymerase. 
     The QM™ (quantitative methylation) assay is an alternative quantitative test for methylation patterns in genomic DNA samples, wherein sequence discrimination occurs at the level of probe hybridization. In this quantitative version, the PCR reaction provides for unbiased amplification in the presence of a fluorescent probe that overlaps a particular putative methylation site. An unbiased control for the amount of input DNA is provided by a reaction in which neither the primers, nor the probe, overlie any CpG dinucleotides. Alternatively, a qualitative test for genomic methylation is achieved by probing the biased PCR pool with either control oligonucleotides that do not cover known methylation sites (a fluorescence-based version of the HeavyMethyl™ and MSP techniques) or with oligonucleotides covering potential methylation sites. 
     The QM™ process can be used with any suitable probe, e.g., “TaqMan®” probes, Lightcycler® probes, in the amplification process. For example, double-stranded genomic DNA is treated with sodium bisulfite and subjected to unbiased primers and the TaqMan® probe. The TaqMan® probe is dual-labeled with fluorescent “reporter” and “quencher” molecules, and is designed to be specific for a relatively high GC content region so that it melts out at about a 10° C. higher temperature in the PCR cycle than the forward or reverse primers. This allows the TaqMan® probe to remain fully hybridized during the PCR annealing/extension step. As the Taq polymerase enzymatically synthesizes a new strand during PCR, it will eventually reach the annealed TaqMan® probe. The Taq polymerase 5′ to 3′ endonuclease activity will then displace the TaqMan® probe by digesting it to release the fluorescent reporter molecule for quantitative detection of its now unquenched signal using a real-time fluorescent detection system. Typical reagents (e.g., as might be found in a typical QM™-based kit) for QM™ analysis may include, but are not limited to: PCR primers for specific loci (e.g., specific genes, markers, regions of genes, regions of markers, bisulfite treated DNA sequence, CpG island, etc.); TaqMan® or Lightcycler® probes; optimized PCR buffers and deoxynucleotides; and Taq polymerase. 
     The Ms-SNuPE™ technique is a quantitative method for assessing methylation differences at specific CpG sites based on bisulfite treatment of DNA, followed by single-nucleotide primer extension (Gonzalgo &amp; Jones, Nucleic Acids Res. 25:2529-2531, 1997). Briefly, genomic DNA is reacted with sodium bisulfite to convert unmethylated cytosine to uracil while leaving 5-methylcytosine unchanged. Amplification of the desired target sequence is then performed using PCR primers specific for bisulfite-converted DNA, and the resulting product is isolated and used as a template for methylation analysis at the CpG site of interest. Small amounts of DNA can be analyzed (e.g., microdissected pathology sections) and it avoids utilization of restriction enzymes for determining the methylation status at CpG sites. 
     Typical reagents (e.g., as might be found in a typical Ms-SNuPE™-based kit) for Ms-SNuPE™ analysis may include, but are not limited to: PCR primers for specific loci (e.g., specific genes, markers, regions of genes, regions of markers, bisulfite treated DNA sequence, CpG island, etc.); optimized PCR buffers and deoxynucleotides; gel extraction kit; positive control primers; Ms-SNuPE™ primers for specific loci; reaction buffer (for the Ms-SNuPE reaction); and labeled nucleotides. Additionally, bisulfite conversion reagents may include: DNA denaturation buffer; sulfonation buffer; DNA recovery reagents or kit (e.g., precipitation, ultrafiltration, affinity column); desulfonation buffer; and DNA recovery components. 
     Reduced Representation Bisulfite Sequencing (RRBS) begins with bisulfite treatment of nucleic acid to convert all unmethylated cytosines to uracil, followed by restriction enzyme digestion (e.g., by an enzyme that recognizes a site including a CG sequence such as MspI) and complete sequencing of fragments after coupling to an adapter ligand. The choice of restriction enzyme enriches the fragments for CpG dense regions, reducing the number of redundant sequences that may map to multiple gene positions during analysis. As such, RRBS reduces the complexity of the nucleic acid sample by selecting a subset (e.g., by size selection using preparative gel electrophoresis) of restriction fragments for sequencing. As opposed to whole-genome bisulfite sequencing, every fragment produced by the restriction enzyme digestion contains DNA methylation information for at least one CpG dinucleotide. As such, RRBS enriches the sample for promoters, CpG islands, and other genomic features with a high frequency of restriction enzyme cut sites in these regions and thus provides an assay to assess the methylation state of one or more genomic loci. 
     A typical protocol for RRBS comprises the steps of digesting a nucleic acid sample with a restriction enzyme such as MspI, filling in overhangs and A-tailing, ligating adaptors, bisulfite conversion, and PCR. See, e.g., et al. (2005) “Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution”  Nat Methods  7: 133-6; Meissner et al. (2005) “Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis”  Nucleic Acids Res.  33: 5868-77. 
     In some embodiments, a quantitative allele-specific real-time target and signal amplification (QuARTS) assay is used to evaluate methylation state. Three reactions sequentially occur in each QuARTS assay, including amplification (reaction 1) and target probe cleavage (reaction 2) in the primary reaction; and FRET cleavage and fluorescent signal generation (reaction 3) in the secondary reaction. When target nucleic acid is amplified with specific primers, a specific detection probe with a flap sequence loosely binds to the amplicon. The presence of the specific invasive oligonucleotide at the target binding site causes a 5′ nuclease, e.g., a FEN-1 endonuclease, to release the flap sequence by cutting between the detection probe and the flap sequence. The flap sequence is complementary to a non-hairpin portion of a corresponding FRET cassette. Accordingly, the flap sequence functions as an invasive oligonucleotide on the FRET cassette and effects a cleavage between the FRET cassette fluorophore and a quencher, which produces a fluorescent signal. The cleavage reaction can cut multiple probes per target and thus release multiple fluorophores per flap, providing exponential signal amplification. QuARTS can detect multiple targets in a single reaction well by using FRET cassettes with different dyes. See, e.g., in Zou et al. (2010) “Sensitive quantification of methylated markers with a novel methylation specific technology”  Clin Chem  56: A199), and U.S. Pat. Nos. 8,361,720; 8,715,937; 8,916,344; and 9,212,392, each of which is incorporated herein by reference for all purposes. 
     The term “bisulfite reagent” refers to a reagent comprising bisulfite, disulfite, hydrogen sulfite, or combinations thereof, useful as disclosed herein to distinguish between methylated and unmethylated CpG dinucleotide sequences. Methods of said treatment are known in the art (e.g., PCT/EP2004/011715 and WO 2013/116375, each of which is incorporated by reference in its entirety). In some embodiments, bisulfite treatment is conducted in the presence of denaturing solvents such as but not limited to n-alkyleneglycol or diethylene glycol dimethyl ether (DME), or in the presence of dioxane or dioxane derivatives. In some embodiments the denaturing solvents are used in concentrations between 1% and 35% (v/v). In some embodiments, the bisulfite reaction is carried out in the presence of scavengers such as but not limited to chromane derivatives, e.g., 6-hydroxy-2,5,7,8,-tetramethylchromane 2-carboxylic acid or trihydroxybenzone acid and derivates thereof, e.g., Gallic acid (see: PCT/EP2004/011715, which is incorporated by reference in its entirety). In certain preferred embodiments, the bisulfite reaction comprises treatment with ammonium hydrogen sulfite, e.g., as described in WO 2013/116375. 
     In some embodiments, fragments of the treated DNA are amplified using sets of primer oligonucleotides according to the present invention (e.g., see Table V) and an amplification enzyme. The amplification of several DNA segments can be carried out simultaneously in one and the same reaction vessel. Typically, the amplification is carried out using a polymerase chain reaction (PCR). Amplicons are typically 100 to 2000 base pairs in length. 
     In some embodiments, the technology relates to assessing the methylation state of combinations of markers comprising a DMR from Tables 1A, 2A, 7A, 8A, and 9 (e.g., DMR Nos. 1-331). In some embodiments, assessing the methylation state of more than one marker increases the specificity and/or sensitivity of a screen or diagnostic for identifying a neoplasm in a subject (e.g., melanoma). 
     In another embodiment, the invention provides a method for converting an oxidized 5-methylcytosine residue in cell-free DNA to a dihydrouracil residue (see, Liu et al., 2019, Nat Biotechnol. 37, pp. 424-429; U.S. Patent Application Publication No. 202000370114). The method involves reaction of an oxidized 5mC residue selected from 5-formylcytosine (5fC), 5-carboxymethylcytosine (5caC), and combinations thereof, with an organic borane. The oxidized 5mC residue may be naturally occurring or, more typically, the result of a prior oxidation of a 5mC or 5hmC residue, e.g., oxidation of 5mC or 5hmC with a TET family enzyme (e.g., TET1, TET2, or TET3), or chemical oxidation of 5 mC or 5hmC, e.g., with potassium perruthenate (KRuO 4 ) or an inorganic peroxo compound or composition such as peroxotungstate (see, e.g., Okamoto et al. (2011)  Chem. Commun.  47:11231-33) and a copper (II) perchlorate/2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) combination (see Matsushita et al. (2017)  Chem. Commun.  53:5756-59). 
     The organic borane may be characterized as a complex of borane and a nitrogen-containing compound selected from nitrogen heterocycles and tertiary amines. The nitrogen heterocycle may be monocyclic, bicyclic, or polycyclic, but is typically monocyclic, in the form of a 5- or 6-membered ring that contains a nitrogen heteroatom and optionally one or more additional heteroatoms selected from N, O, and S. The nitrogen heterocycle may be aromatic or alicyclic. Preferred nitrogen heterocycles herein include 2-pyrroline, 2H-pyrrole, 1H-pyrrole, pyrazolidine, imidazolidine, 2-pyrazoline, 2-imidazoline, pyrazole, imidazole, 1,2,4-triazole, 1,2,4-triazole, pyridazine, pyrimidine, pyrazine, 1,2,4-triazine, and 1,3,5-triazine, any of which may be unsubstituted or substituted with one or more non-hydrogen substituents. Typical non-hydrogen substituents are alkyl groups, particularly lower alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, and the like. Exemplary compounds include pyridine borane, 2-methylpyridine borane (also referred to as 2-picoline borane), and 5-ethyl-2-pyridine. 
     The reaction of the organic borane with the oxidized 5mC residue in cell-free DNA is advantageous insofar as non-toxic reagents and mild reaction conditions can be employed; there is no need for any bisulfite, nor for any other potentially DNA-degrading reagents. Furthermore, conversion of an oxidized 5mC residue to dihydrouracil with the organic borane can be carried out without need for isolation of any intermediates, in a “one-pot” or “one-tube” reaction. This is quite significant, since the conversion involves multiple steps, i.e., (1) reduction of the alkene bond linking C-4 and C-5 in the oxidized 5mC, (2) deamination, and (3) either decarboxylation, if the oxidized 5mC is 5caC, or deformylation, if the oxidized 5mC is 5fC. 
     In addition to a method for converting an oxidized 5-methylcytosine residue in cell-free DNA to a dihydrouracil residue, the invention also provides a reaction mixture related to the aforementioned method. The reaction mixture comprises a sample of cell-free DNA containing at least one oxidized 5-methylcytosine residue selected from 5caC, 5fC, and combinations thereof, and an organic borane effective to reduce, deaminate, and either decarboxylate or deformylate the at least one oxidized 5-methylcytosine residue. The organic borane is a complex of borane and a nitrogen-containing compound selected from nitrogen heterocycles and tertiary amines, as explained above. In a preferred embodiment, the reaction mixture is substantially free of bisulfite, meaning substantially free of bisulfite ion and bisulfite salts. Ideally, the reaction mixture contains no bisulfite. 
     In a related aspect of the invention, a kit is provided for converting 5mC residues in cell-free DNA to dihydrouracil residues, where the kit includes a reagent for blocking 5hmC residues, a reagent for oxidizing 5mC residues beyond hydroxymethylation to provide oxidized 5mC residues, and an organic borane effective to reduce, deaminate, and either decarboxylate or deformylate the oxidized 5mC residues. The kit may also include instructions for using the components to carry out the above-described method. 
     In another embodiment, a method is provided that makes use of the above-described oxidation reaction. The method enables detecting the presence and location of 5-methylcytosine residues in cell-free DNA, and comprises the following steps: 
     (a) modifying 5hmC residues in fragmented, adapter-ligated cell-free DNA to provide an affinity tag thereon, wherein the affinity tag enables removal of modified 5hmC-containing DNA from the cell-free DNA; 
     (b) removing the modified 5hmC-containing DNA from the cell-free DNA, leaving DNA containing unmodified 5mC residues; 
     (c) oxidizing the unmodified 5mC residues to give DNA containing oxidized 5mC residues selected from 5caC, 5fC, and combinations thereof; 
     (d) contacting the DNA containing oxidized 5mC residues with an organic borane effective to reduce, deaminate, and either decarboxylate or deformylate the oxidized 5mC residues, thereby providing DNA containing dihydrouracil residues in place of the oxidized 5mC residues; 
     (e) amplifying and sequencing the DNA containing dihydrouracil residues; 
     (f) determining a 5-methylation pattern from the sequencing results in (e). 
     The cell-free DNA is extracted from a body sample from a subject, where the body sample is typically whole blood, plasma, or serum, most typically plasma, but the sample may also be urine, saliva, mucosal excretions, sputum, stool, or tears. In some embodiments, the cell-free DNA is derived from a tumor. In other embodiments, the cell-free DNA is from a patient with a disease or other pathogenic condition. The cell-free DNA may or may not derive from a tumor. In step (a), it should be noted that the cell-free DNA in which 5hmC residues are to be modified is in purified, fragmented form, and adapter-ligated. DNA purification in this context can be carried out using any suitable method known to those of ordinary skill in the art and/or described in the pertinent literature, and, while cell-free DNA can itself be highly fragmented, further fragmentation may occasionally be desirable, as described, for example, in U.S. Patent Publication No. 2017/0253924. The cell-free DNA fragments are generally in the size range of about 20 nucleotides to about 500 nucleotides, more typically in the range of about 20 nucleotides to about 250 nucleotides. The purified cell-free DNA fragments that are modified in step (a) have been end-repaired using conventional means (e.g., a restriction enzyme) so that the fragments have a blunt end at each 3′ and 5′ terminus. In a preferred method, as described in WO 2017/176630, the blunted fragments have also been provided with a 3′ overhang comprising a single adenine residue using a polymerase such as Taq polymerase. This facilitates subsequent ligation of a selected universal adapter, i.e., an adapter such as a Y-adapter or a hairpin adapter that ligates to both ends of the cell-free DNA fragments and contains at least one molecular barcode. Use of adapters also enables selective PCR enrichment of adapter-ligated DNA fragments. 
     In step (a), then, the “purified, fragmented cell-free DNA” comprises adapter-ligated DNA fragments. Modification of 5hmC residues in these cell-free DNA fragments with an affinity tag, as specified in step (a), is done so as to enable subsequent removal of the modified 5hmC-containing DNA from the cell-free DNA. In one embodiment, the affinity tag comprises a biotin moiety, such as biotin, desthiobiotin, oxybiotin, 2-iminobiotin, diaminobiotin, biotin sulfoxide, biocytin, or the like. Use of a biotin moiety as the affinity tag allows for facile removal with streptavidin, e.g., streptavidin beads, magnetic streptavidin beads, etc. 
     Tagging 5hmC residues with a biotin moiety or other affinity tag is accomplished by covalent attachment of a chemoselective group to 5hmC residues in the DNA fragments, where the chemoselective group is capable of undergoing reaction with a functionalized affinity tag so as to link the affinity tag to the 5hmC residues. In one embodiment, the chemoselective group is UDP glucose-6-azide, which undergoes a spontaneous 1,3-cycloaddition reaction with an alkyne-functionalized biotin moiety, as described in Robertson et al. (2011)  Biochem. Biophys. Res. Comm.  411(1):40-3, U.S. Pat. No. 8,741,567, and WO 2017/176630. Addition of an alkyne-functionalized biotin-moiety thus results in covalent attachment of the biotin moiety to each 5hmC residue. 
     The affinity-tagged DNA fragments can then be pulled down in step (b) using, in one embodiment, streptavidin, in the form of streptavidin beads, magnetic streptavidin beads, or the like, and set aside for later analysis, if so desired. The supernatant remaining after removal of the affinity-tagged fragments contains DNA with unmodified 5mC residues and no 5hmC residues. 
     In step (c), the unmodified 5mC residues are oxidized to provide 5caC residues and/or 5fC residues, using any suitable means. The oxidizing agent is selected to oxidize 5mC residues beyond hydroxymethylation, i.e., to provide 5caC and/or 5fC residues. Oxidation may be carried out enzymatically, using a catalytically active TET family enzyme. A “TET family enzyme” or a “TET enzyme” as those terms are used herein refer to a catalytically active “TET family protein” or a “TET catalytically active fragment” as defined in U.S. Pat. No. 9,115,386, the disclosure of which is incorporated by reference herein. A preferred TET enzyme in this context is TET2; see Ito et al. (2011)  Science  333(6047):1300-1303. Oxidation may also be carried out chemically, as described in the preceding section, using a chemical oxidizing agent. Examples of suitable oxidizing agent include, without limitation: a perruthenate anion in the form of an inorganic or organic perruthenate salt, including metal perruthenates such as potassium perruthenate (KRuO 4 ), tetraalkylammonium perruthenates such as tetrapropylammonium perruthenate (TPAP) and tetrabutylammonium perruthenate (TBAP), and polymer supported perruthenate (PSP); and inorganic peroxo compounds and compositions such as peroxotungstate or a copper (II) perchlorate/TEMPO combination. It is unnecessary at this point to separate 5fC-containing fragments from 5caC-containing fragments, insofar as in the next step of the process, step (e) converts both 5fC residues and 5caC residues to dihydrouracil (DHU). 
     In some embodiments, 5-hydroxymethylcytosine residues are blocked with (3-glucosyltransferase (β3GT), while 5-methylcytosine residues are oxidized with a TET enzyme effective to provide a mixture of 5-formylcytosine and 5-carboxymethylcytosine. The mixture containing both of these oxidized species can be reacted with 2-picoline borane or another organic borane to give dihydrouracil. In a variation on this embodiment, 5hmC-containing fragments are not removed in step (b). Rather, “TET-Assisted Picoline Borane Sequencing (TAPS),” 5mC-containing fragments and 5hmC-containing fragments are together enzymatically oxidized to provide 5fC- and 5caC-containing fragments. Reaction with 2-picoline borane results in DHU residues wherever 5mC and 5hmC residues were originally present. “Chemical Assisted Picoline Borane Sequencing (CAPS),” involves selective oxidation of 5hmC-containing fragments with potassium perruthenate, leaving 5mC residues unchanged. 
     There are numerous advantages to the method of this embodiment: bisulfite is unnecessary, nontoxic reagents and reactants are employed; and the process proceeds under mild conditions. In addition, the entire process can be performed in a single tube, without need for isolation of any intermediates. 
     In a related embodiment, the above method includes a further step: (g) identifying a hydroxymethylation pattern in the 5hmC-containing DNA removed from the cell-free DNA in step (b). This can be carried out using the techniques described in detail in WO 2017/176630. The process can be carried out without removal or isolation of intermediates in a one-tube method. For example, initially, cell-free DNA fragments, preferably adapter-ligated DNA fragments, are subjected to functionalization with OGT-catalyzed uridine diphosphoglucose 6-azide, followed by biotinylation via the chemoselective azide groups. This procedure results in covalently attached biotin at each 5hmC site. In a next step, the biotinylated strands and strands containing unmodified (native) 5mC are pulled down simultaneously for further processing. The native 5mC-containing strands are pulled down using an anti-5mC antibody or a methyl-CpG-binding domain (MBD) protein, as is known in the art. Then, with the 5hmC residues blocked, the unmodified 5mC residues are selectively oxidized using any suitable technique for converting 5mC to 5fC and/or 5caC, as described elsewhere herein. 
     The fragments obtained by means of the amplification can carry a directly or indirectly detectable label. In some embodiments, the labels are fluorescent labels, radionuclides, or detachable molecule fragments having a typical mass that can be detected in a mass spectrometer. Where said labels are mass labels, some embodiments provide that the labeled amplicons have a single positive or negative net charge, allowing for better delectability in the mass spectrometer. The detection may be carried out and visualized by means of, e.g., matrix assisted laser desorption/ionization mass spectrometry (MALDI) or using electron spray mass spectrometry (ESI). 
     Methods for isolating DNA suitable for these assay technologies are known in the art. In particular, some embodiments comprise isolation of nucleic acids as described in U.S. patent application Ser. No. 13/470,251 (“Isolation of Nucleic Acids”), incorporated herein by reference in its entirety. 
     In some embodiments, the markers described herein find use in QUARTS assays performed on stool samples. In some embodiments, methods for producing DNA samples and, in particular, to methods for producing DNA samples that comprise highly purified, low-abundance nucleic acids in a small volume (e.g., less than 100, less than 60 microliters) and that are substantially and/or effectively free of substances that inhibit assays used to test the DNA samples (e.g., PCR, INVADER, QuARTS assays, etc.) are provided. Such DNA samples find use in diagnostic assays that qualitatively detect the presence of, or quantitatively measure the activity, expression, or amount of, a gene, a gene variant (e.g., an allele), or a gene modification (e.g., methylation) present in a sample taken from a patient. For example, some cancers are correlated with the presence of particular mutant alleles or particular methylation states, and thus detecting and/or quantifying such mutant alleles or methylation states has predictive value in the diagnosis and treatment of cancer. 
     Many valuable genetic markers are present in extremely low amounts in samples and many of the events that produce such markers are rare. Consequently, even sensitive detection methods such as PCR require a large amount of DNA to provide enough of a low-abundance target to meet or supersede the detection threshold of the assay. Moreover, the presence of even low amounts of inhibitory substances compromise the accuracy and precision of these assays directed to detecting such low amounts of a target. Accordingly, provided herein are methods providing the requisite management of volume and concentration to produce such DNA samples. 
     In some embodiments, the sample comprises stool, tissue sample (e.g., skin tissue), an organ secretion, CSF, saliva, blood, or urine. In some embodiments, the subject is human. Such samples can be obtained by any number of means known in the art, such as will be apparent to the skilled person. Cell free or substantially cell free samples can be obtained by subjecting the sample to various techniques known to those of skill in the art which include, but are not limited to, centrifugation and filtration. Although it is generally preferred that no invasive techniques are used to obtain the sample, it still may be preferable to obtain samples such as tissue homogenates, tissue sections, and biopsy specimens. The technology is not limited in the methods used to prepare the samples and provide a nucleic acid for testing. For example, in some embodiments, a DNA is isolated from a stool sample or from blood or from a plasma sample using direct gene capture, e.g., as detailed in U.S. Pat. Nos. 8,808,990 and 9,169,511, and in WO 2012/155072, or by a related method. 
     The analysis of markers can be carried out separately or simultaneously with additional markers within one test sample. For example, several markers can be combined into one test for efficient processing of multiple samples and for potentially providing greater diagnostic and/or prognostic accuracy. In addition, one skilled in the art would recognize the value of testing multiple samples (for example, at successive time points) from the same subject. Such testing of serial samples can allow the identification of changes in marker methylation states over time. Changes in methylation state, as well as the absence of change in methylation state, can provide useful information about the disease status that includes, but is not limited to, identifying the approximate time from onset of the event, the presence and amount of salvageable tissue, the appropriateness of drug therapies, the effectiveness of various therapies, and identification of the subject&#39;s outcome, including risk of future events. The analysis of biomarkers can be carried out in a variety of physical formats. For example, the use of microtiter plates or automation can be used to facilitate the processing of large numbers of test samples. Alternatively, single sample formats could be developed to facilitate immediate treatment and diagnosis in a timely fashion, for example, in ambulatory transport or emergency room settings. 
     It is contemplated that embodiments of the technology are provided in the form of a kit. The kits comprise embodiments of the compositions, devices, apparatuses, etc. described herein, and instructions for use of the kit. Such instructions describe appropriate methods for preparing an analyte from a sample, e.g., for collecting a sample and preparing a nucleic acid from the sample. Individual components of the kit are packaged in appropriate containers and packaging (e.g., vials, boxes, blister packs, ampules, jars, bottles, tubes, and the like) and the components are packaged together in an appropriate container (e.g., a box or boxes) for convenient storage, shipping, and/or use by the user of the kit. It is understood that liquid components (e.g., a buffer) may be provided in a lyophilized form to be reconstituted by the user. Kits may include a control or reference for assessing, validating, and/or assuring the performance of the kit. For example, a kit for assaying the amount of a nucleic acid present in a sample may include a control comprising a known concentration of the same or another nucleic acid for comparison and, in some embodiments, a detection reagent (e.g., a primer) specific for the control nucleic acid. The kits are appropriate for use in a clinical setting and, in some embodiments, for use in a user&#39;s home. The components of a kit, in some embodiments, provide the functionalities of a system for preparing a nucleic acid solution from a sample. In some embodiments, certain components of the system are provided by the user. 
     Various cancers are predicted by various combinations of markers, e.g., as identified by statistical techniques related to specificity and sensitivity of prediction. The technology provides methods for identifying predictive combinations and validated predictive combinations for some cancers. 
     Methods 
     In some embodiments of the technology, methods are provided that comprise the following steps:
         1) contacting a nucleic acid (e.g., genomic DNA, e.g., isolated from body fluids such as blood or plasma, fine needle aspirate, deep tissue, or skin tissue) obtained from the subject with at least one reagent or series of reagents that distinguishes between methylated and non-methylated CpG dinucleotides within at least one marker comprising a DMR (e.g., DMR 1-331 e.g., as provided in Tables 1A, 2A, 7A, 8A, and 9) and   2) detecting melanoma, metastatic melanoma, or primary cutaneous melanoma (e.g., afforded with a sensitivity of greater than or equal to 80% and a specificity of greater than or equal to 80%).       

     In some embodiments of the technology, methods are provided that comprise the following steps:
         1) contacting a nucleic acid (e.g., genomic DNA, e.g., isolated from body fluids such as blood or plasma, fine needle aspirate, deep tissue, or skin tissue) obtained from the subject with at least one reagent or series of reagents that distinguishes between methylated and non-methylated CpG dinucleotides within at least one marker selected from a chromosomal region having an annotation selected from the group consisting of c10orf55, c2orf82, FOXL2NB, CLIC5, FAM174B_B, FLJ22536_A, FOXP1, GALNT3_A, HOPX, HOXA9_A, ITPKA, KCNQ4_A, LMX1B, MAX.chr1.110627096-110627364, MAX.chr10.22541869-22541953, MAX.chr10.62492690-62492812, MAX.chr13.29106812-29106917, MAX.chr17.73073682-73073830, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, MAX.chr5.42992655-42992768, MAX.chr5.60921627-60921853, MAX.chr6.29521537-29521696, MAX.chr7.155259597-155259763, ME3, MIR155HG, NFATC2_A, OCA2, OXT, RNF207_A, RUNX2, SIX4_A, STX16, TAL1, TMEM30B, and TSPAN33, and   2) detecting melanoma (e.g., afforded with a sensitivity of greater than or equal to 80% and a specificity of greater than or equal to 80%).       

     In some embodiments of the technology, methods are provided that comprise the following steps:
         1) contacting a nucleic acid (e.g., genomic DNA, e.g., isolated from body fluids such as blood or plasma, fine needle aspirate, deep tissue, or skin tissue) obtained from the subject with at least one reagent or series of reagents that distinguishes between methylated and non-methylated CpG dinucleotides within at least one marker selected from a chromosomal region having an annotation selected from the group consisting of MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-73073830, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22541874-22541948, ME3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763, and   2) detecting melanoma (e.g., afforded with a sensitivity of greater than or equal to 80% and a specificity of greater than or equal to 80%).       

     In some embodiments of the technology, methods are provided that comprise the following steps:
         1) contacting a nucleic acid (e.g., genomic DNA, e.g., isolated from body fluids such as blood or plasma, fine needle aspirate, deep tissue, or skin tissue) obtained from the subject with at least one reagent or series of reagents that distinguishes between methylated and non-methylated CpG dinucleotides within at least one marker selected from a chromosomal region having an annotation selected from the group consisting of AGRN, BMP8B, c17orf46_B, c17orf57_B, C2CD4A, C2CD4D_B, CDYL, CMAH, DENND2D, DLEU2, DSCR6, FLJ22536_B, FLJ45983, FOXF2, GALNT3_B, HCG4P6, HOXA9_B, KIFC2, LDLRAD2, LY75, LYL1, LYN, MAPK13, MAX.chr1.1072486-1072508, MAX.chr1.32237693-32237785, MAX.chr10.62492374-62492793, MAX.chr11.14926535-14926715, MAX.chr16.54970444-54970469, MAX.chr19.16439332-16439390, MAX.chr20.21080670-21081280, MAX.chr4.113432264-113432298, MAX.chr7.129425668-129425719, MAX.chr8.82543163-82543213, PARP15, PRKAG2, PROC_A, PROM1_B, PTGER4_A, PTP4A3, SDCCAG8, SH3PXD2A, SLC2A2, SLC35D3, TBR1, TBX2, TCP11, TFAP2A, and TRIM73, and   2) detecting melanoma (e.g., afforded with a sensitivity of greater than or equal to 80% and a specificity of greater than or equal to 80%).       

     In some embodiments of the technology, methods are provided that comprise the following steps:
         1) contacting a nucleic acid (e.g., genomic DNA, e.g., isolated from body fluids such as blood or plasma, fine needle aspirate, deep tissue, or skin tissue) obtained from the subject with at least one reagent or series of reagents that distinguishes between methylated and non-methylated CpG dinucleotides within at least one marker selected from a chromosomal region having an annotation selected from the group consisting of MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763, FOXL2NB, and HOXA9_A, and   2) detecting metastatic melanoma (e.g., afforded with a sensitivity of greater than or equal to 80% and a specificity of greater than or equal to 80%).       

     In some embodiments of the technology, methods are provided that comprise the following steps:
         1) contacting a nucleic acid (e.g., genomic DNA, e.g., isolated from body fluids such as blood or plasma, fine needle aspirate, deep tissue, or skin tissue) obtained from the subject with at least one reagent or series of reagents that distinguishes between methylated and non-methylated CpG dinucleotides within at least one marker selected from a chromosomal region having an annotation selected from the group consisting of MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-73073830, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22541874-22541948, ME3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763, and   2) detecting metastatic melanoma (e.g., afforded with a sensitivity of greater than or equal to 80% and a specificity of greater than or equal to 80%).       

     In some embodiments of the technology, methods are provided that comprise the following steps: 
     1) measuring a methylation level for one or more genes in a biological sample of a human individual through treating genomic DNA in the biological sample with a reagent that modifies DNA in a methylation-specific manner (e.g., wherein the reagent is a bisulfite reagent, a methylation-sensitive restriction enzyme, or a methylation-dependent restriction enzyme), wherein the one or more genes is selected from one of the following groups:
         c10orf55, c2orf82, FOXL2NB, CLIC5, FAM174B_B, FLJ22536_A, FOXP1, GALNT3_A, HOPX, HOXA9_A, ITPKA, KCNQ4_A, LMX1B, MAX.chr1.110627096-110627364, MAX.chr10.22541869-22541953, MAX.chr10.62492690-62492812, MAX.chr13.29106812-29106917, MAX.chr17.73073682-73073830, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, MAX.chr5.42992655-42992768, MAX.chr5.60921627-60921853, MAX.chr6.29521537-29521696, MAX.chr7.155259597-155259763, ME3, MIR155HG, NFATC2_A, OCA2, OXT, RNF207_A, RUNX2, SIX4_A, STX16, TAL1, TMEM30B, and TSPAN33 (see, Tables 4, 5A, 5B and 6; Example I);   MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-73073830, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22541874-22541948, ME3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763 (see, Tables 9, 10 and 11);   AGRN, BMP8B, c17orf46_B, c17orf57_B, C2CD4A, C2CD4D_B, CDYL, CMAH, DENND2D, DLEU2, DSCR6, FLJ22536_B, FLJ45983, FOXF2, GALNT3_B, HCG4P6, HOXA9_B, KIFC2, LDLRAD2, LY75, LYL1, LYN, MAPK13, MAX.chr1.1072486-1072508, MAX.chr1.32237693-32237785, MAX.chr10.62492374-62492793, MAX.chr11.14926535-14926715, MAX.chr16.54970444-54970469, MAX.chr19.16439332-16439390, MAX.chr20.21080670-21081280, MAX.chr4.113432264-113432298, MAX.chr7.129425668-129425719, MAX.chr8.82543163-82543213, PARP15, PRKAG2, PROC_A, PROM1_B, PTGER4_A, PTP4A3, SDCCAG8, SH3PXD2A, SLC2A2, SLC35D3, TBR1, TBX2, TCP11, TFAP2A, and TRIM73 (see, Tables 7A and 7B; Example II);   One or more markers recited in Tables 8A and 8B (Example II); and   MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763, FOXL2NB, and HOXA9_A (see, Table 5C, Example I);       

     2) amplifying the treated genomic DNA using a set of primers for the selected one or more genes; and 
     3) determining the methylation level of the one or more genes by polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nuclease, mass-based separation, and target capture. 
     In some embodiments of the technology, methods are provided that comprise the following steps: 
     1) measuring an amount of at least one methylated marker gene in DNA from the sample, wherein the one or more genes is selected from one of the following groups:
         c10orf55, c2orf82, FOXL2NB, CLIC5, FAM174B_B, FLJ22536_A, FOXP1, GALNT3_A, HOPX, HOXA9_A, ITPKA, KCNQ4_A, LMX1B, MAX.chr1.110627096-110627364, MAX.chr10.22541869-22541953, MAX.chr10.62492690-62492812, MAX.chr13.29106812-29106917, MAX.chr17.73073682-73073830, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, MAX.chr5.42992655-42992768, MAX.chr5.60921627-60921853, MAX.chr6.29521537-29521696, MAX.chr7.155259597-155259763, ME3, MIR155HG, NFATC2_A, OCA2, OXT, RNF207_A, RUNX2, SIX4_A, STX16, TAL1, TMEM30B, and TSPAN33 (see, Tables 4, 5A, 5B and 6; Example I;   MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-73073830, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22541874-22541948, ME3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763 (see, Tables 9, 10 and 11);   AGRN, BMP8B, c17orf46_B, c17orf57_B, C2CD4A, C2CD4D_B, CDYL, CMAH, DENND2D, DLEU2, DSCR6, FLJ22536_B, FLJ45983, FOXF2, GALNT3_B, HCG4P6, HOXA9_B, KIFC2, LDLRAD2, LY75, LYL1, LYN, MAPK13, MAX.chr1.1072486-1072508, MAX.chr1.32237693-32237785, MAX.chr10.62492374-62492793, MAX.chr11.14926535-14926715, MAX.chr16.54970444-54970469, MAX.chr19.16439332-16439390, MAX.chr20.21080670-21081280, MAX.chr4.113432264-113432298, MAX.chr7.129425668-129425719, MAX.chr8.82543163-82543213, PARP15, PRKAG2, PROC_A, PROM1_B, PTGER4_A, PTP4A3, SDCCAG8, SH3PXD2A, SLC2A2, SLC35D3, TBR1, TBX2, TCP11, TFAP2A, and TRIM73 (see, Tables 7A and 7B; Example II);   One or more markers recited in Tables 8A and 8B (Example II); and   MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763, FOXL2NB, and HOXA9_A (see, Table 5C, Example I);       

     2) measuring the amount of at least one reference marker in the DNA; and 
     3) calculating a value for the amount of the at least one methylated marker gene measured in the DNA as a percentage of the amount of the reference marker gene measured in the DNA, wherein the value indicates the amount of the at least one methylated marker DNA measured in the sample. 
     In some embodiments of the technology, methods are provided that comprise the following steps: 
     1) measuring a methylation level of a CpG site for one or more genes in a biological sample of a human individual through treating genomic DNA in the biological sample with bisulfite a reagent capable of modifying DNA in a methylation-specific manner (e.g., a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and a bisulfite reagent); 
     2) amplifying the modified genomic DNA using a set of primers for the selected one or more genes; and 
     3) determining the methylation level of the CpG site by methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, or bisulfite genomic sequencing PCR;
         wherein the one or more genes is selected from one of the following groups:   c10orf55, c2orf82, FOXL2NB, CLIC5, FAM174B_B, FLJ22536_A, FOXP1, GALNT3_A, HOPX, HOXA9_A, ITPKA, KCNQ4_A, LMX1B, MAX.chr1.110627096-110627364, MAX.chr10.22541869-22541953, MAX.chr10.62492690-62492812, MAX.chr13.29106812-29106917, MAX.chr17.73073682-73073830, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, MAX.chr5.42992655-42992768, MAX.chr5.60921627-60921853, MAX.chr6.29521537-29521696, MAX.chr7.155259597-155259763, ME3, MIR155HG, NFATC2_A, OCA2, OXT, RNF207_A, RUNX2, SIX4_A, STX16, TAL1, TMEM30B, and TSPAN33 (see, Tables 4, 5A, 5B and 6; Example I;   MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-73073830, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22541874-22541948, ME3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763 (see, Tables 9, 10 and 11);   AGRN, BMP8B, c17orf46_B, c17orf57_B, C2CD4A, C2CD4D_B, CDYL, CMAH, DENND2D, DLEU2, DSCR6, FLJ22536_B, FLJ45983, FOXF2, GALNT3_B, HCG4P6, HOXA9_B, KIFC2, LDLRAD2, LY75, LYL1, LYN, MAPK13, MAX.chr1.1072486-1072508, MAX.chr1.32237693-32237785, MAX.chr10.62492374-62492793, MAX.chr11.14926535-14926715, MAX.chr16.54970444-54970469, MAX.chr19.16439332-16439390, MAX.chr20.21080670-21081280, MAX.chr4.113432264-113432298, MAX.chr7.129425668-129425719, MAX.chr8.82543163-82543213, PARP15, PRKAG2, PROC_A, PROM1_B, PTGER4_A, PTP4A3, SDCCAG8, SH3PXD2A, SLC2A2, SLC35D3, TBR1, TBX2, TCP11, TFAP2A, and TRIM73 (see, Tables 7A and 7B; Example II);   One or more markers recited in Tables 8A and 8B (Example II); and   MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763, FOXL2NB, and HOXA9_A (see, Table 5C, Example I).       

     Within any of such methods, determining the methylation level for any of such markers is accomplished with the primers recited in Table 3. 
     Preferably, the sensitivity for such methods is from about 70% to about 100%, or from about 80% to about 90%, or from about 80% to about 85%. Preferably, the specificity is from about 70% to about 100%, or from about 80% to about 90%, or from about 80% to about 85%. 
     Genomic DNA may be isolated by any means, including the use of commercially available kits. Briefly, wherein the DNA of interest is encapsulated in by a cellular membrane the biological sample must be disrupted and lysed by enzymatic, chemical or mechanical means. The DNA solution may then be cleared of proteins and other contaminants, e.g., by digestion with proteinase K. The genomic DNA is then recovered from the solution. This may be carried out by means of a variety of methods including salting out, organic extraction, or binding of the DNA to a solid phase support. The choice of method will be affected by several factors including time, expense, and required quantity of DNA. All clinical sample types comprising neoplastic matter or pre-neoplastic matter are suitable for use in the present method, e.g., cell lines, histological slides, biopsies, paraffin-embedded tissue, body fluids, stool, skin tissue, lymphatic tissue or aspirate, brain, lung, liver, bone or other deep organ tissue, colonic effluent, urine, blood plasma, blood serum, whole blood, isolated blood cells, cells isolated from the blood, and combinations thereof. 
     The technology is not limited in the methods used to prepare the samples and provide a nucleic acid for testing. For example, in some embodiments, a DNA is isolated from a skin tissue sample or a stool sample or from blood or from a plasma sample using direct gene capture, e.g., as detailed in U.S. Pat. Appl. Ser. No. 61/485,386 or by a related method. 
     The genomic DNA sample is then treated with at least one reagent, or series of reagents, that distinguishes between methylated and non-methylated CpG dinucleotides within at least one marker comprising a DMR (e.g., DMR 1-331, e.g., as provided by Tables 1A, 2A, 7A, 8A, and 9). 
     In some embodiments, the reagent converts cytosine bases which are unmethylated at the 5′-position to uracil, thymine, or another base which is dissimilar to cytosine in terms of hybridization behavior. However in some embodiments, the reagent may be a methylation sensitive restriction enzyme. 
     In some embodiments, the genomic DNA sample is treated in such a manner that cytosine bases that are unmethylated at the 5′ position are converted to uracil, thymine, or another base that is dissimilar to cytosine in terms of hybridization behavior. In some embodiments, this treatment is carried out with bisulfite (hydrogen sulfite, disulfite) followed by alkaline hydrolysis. 
     The treated nucleic acid is then analyzed to determine the methylation state of the target gene sequences (at least one gene, genomic sequence, or nucleotide from a marker comprising a DMR, e.g., at least one DMR chosen from DMR 1-331, e.g., as provided in Tables 1A, 2A, 7A, 8A, and 9). The method of analysis may be selected from those known in the art, including those listed herein, e.g., QuARTS and MSP as described herein. 
     Aberrant methylation, more specifically hypermethylation of a marker comprising a DMR (e.g., DMR 1-331, e.g., as provided by Tables 1A, 2A, 7A, 8A, and 9) is associated with melanoma. 
     The technology relates to the analysis of any sample associated with melanoma. For example, in some embodiments the sample comprises a tissue (e.g., skin tissue) and/or biological fluid obtained from a patient. In some embodiments, the sample comprises a secretion. In some embodiments, the sample comprises blood, serum, plasma, gastric secretions, pancreatic juice, a gastrointestinal biopsy sample, microdissected cells from a skin tissue biopsy, deep tissue biopsy, fine needle aspirate, and/or cells recovered from stool. In some embodiments, the sample comprises skin tissue. In some embodiments, the subject is human. The sample may include cells, secretions, or tissues from the ovary, breast, liver, bile ducts, pancreas, stomach, colon, skin, rectum, esophagus, small intestine, appendix, duodenum, polyps, gall bladder, anus, lymph nodes, brain, bone, and/or peritoneum. In some embodiments, the sample comprises cellular fluid, ascites, urine, feces, pancreatic fluid, fluid obtained during endoscopy, blood, mucus, or saliva. In some embodiments, the sample is a stool sample. In some embodiments, skin cells are collected using adhesive tape or other adhesive surfaces. 
     Such samples can be obtained by any number of means known in the art, such as will be apparent to the skilled person. For instance, urine and fecal samples are easily attainable, while blood, ascites, serum, or pancreatic fluid samples can be obtained parenterally by using a needle and syringe, for instance. Cell free or substantially cell free samples can be obtained by subjecting the sample to various techniques known to those of skill in the art which include, but are not limited to, centrifugation and filtration. Although it is generally preferred that no invasive techniques are used to obtain the sample, it still may be preferable to obtain samples such as tissue homogenates, tissue sections, and biopsy specimens 
     In some embodiments, the technology relates to a method for treating a patient (e.g., a patient with melanoma) (e.g., a patient with one or more of metastatic melanoma, primary cutaneous melanoma), the method comprising determining the methylation state of one or more DMR as provided herein and administering a treatment to the patient based on the results of determining the methylation state. The treatment may be administration of a pharmaceutical compound, a vaccine, an immunotherapy, performing a surgery, imaging the patient, performing another test. Preferably, said use is in a method of clinical screening, a method of prognosis assessment, a method of monitoring the results of therapy, a method to identify patients most likely to respond to a particular therapeutic treatment, a method of imaging a patient or subject, and a method for drug screening and development. 
     In some embodiments of the technology, a method for diagnosing melanoma in a subject is provided. The terms “diagnosing” and “diagnosis” as used herein refer to methods by which the skilled artisan can estimate and even determine whether or not a subject is suffering from a given disease or condition or may develop a given disease or condition in the future. The skilled artisan often makes a diagnosis on the basis of one or more diagnostic indicators, such as for example a biomarker (e.g., a DMR as disclosed herein), the methylation state of which is indicative of the presence, severity, or absence of the condition. 
     Along with diagnosis, clinical cancer prognosis relates to determining the aggressiveness of the cancer and the likelihood of tumor recurrence to plan the most effective therapy. If a more accurate prognosis can be made or even a potential risk for developing the cancer can be assessed, appropriate therapy, and in some instances less severe therapy for the patient can be chosen. Assessment (e.g., determining methylation state) of cancer biomarkers is useful to separate subjects with good prognosis and/or low risk of developing cancer who will need no therapy or limited therapy from those more likely to develop cancer or suffer a recurrence of cancer who might benefit from more intensive treatments. 
     As such, “making a diagnosis” or “diagnosing”, as used herein, is further inclusive of determining a risk of developing cancer or determining a prognosis, which can provide for predicting a clinical outcome (with or without medical treatment), selecting an appropriate treatment (or whether treatment would be effective), or monitoring a current treatment and potentially changing the treatment, based on the measure of the diagnostic biomarkers (e.g., DMR) disclosed herein. Further, in some embodiments of the presently disclosed subject matter, multiple determination of the biomarkers over time can be made to facilitate diagnosis and/or prognosis. A temporal change in the biomarker can be used to predict a clinical outcome, monitor the progression of melanoma, and/or monitor the efficacy of appropriate therapies directed against the cancer. In such an embodiment for example, one might expect to see a change in the methylation state of one or more biomarkers (e.g., DMR) disclosed herein (and potentially one or more additional biomarker(s), if monitored) in a biological sample over time during the course of an effective therapy. 
     The presently disclosed subject matter further provides in some embodiments a method for determining whether to initiate or continue prophylaxis or treatment of a cancer in a subject. In some embodiments, the method comprises providing a series of biological samples over a time period from the subject; analyzing the series of biological samples to determine a methylation state of at least one biomarker disclosed herein in each of the biological samples; and comparing any measurable change in the methylation states of one or more of the biomarkers in each of the biological samples. Any changes in the methylation states of biomarkers over the time period can be used to predict risk of developing cancer, predict clinical outcome, determine whether to initiate or continue the prophylaxis or therapy of the cancer, and whether a current therapy is effectively treating the cancer. For example, a first time point can be selected prior to initiation of a treatment and a second time point can be selected at some time after initiation of the treatment. Methylation states can be measured in each of the samples taken from different time points and qualitative and/or quantitative differences noted. A change in the methylation states of the biomarker levels from the different samples can be correlated with melanoma risk, prognosis, determining treatment efficacy, and/or progression of the cancer in the subject. 
     In preferred embodiments, the methods and compositions of the invention are for treatment or diagnosis of disease at an early stage, for example, before symptoms of the disease appear. In some embodiments, the methods and compositions of the invention are for treatment or diagnosis of disease at a clinical stage. 
     As noted, in some embodiments, multiple determinations of one or more diagnostic or prognostic biomarkers can be made, and a temporal change in the marker can be used to determine a diagnosis or prognosis. For example, a diagnostic marker can be determined at an initial time, and again at a second time. In such embodiments, an increase in the marker from the initial time to the second time can be diagnostic of a particular type or severity of cancer, or a given prognosis. Likewise, a decrease in the marker from the initial time to the second time can be indicative of a particular type or severity of cancer, or a given prognosis. Furthermore, the degree of change of one or more markers can be related to the severity of the cancer and future adverse events. The skilled artisan will understand that, while in certain embodiments comparative measurements can be made of the same biomarker at multiple time points, one can also measure a given biomarker at one time point, and a second biomarker at a second time point, and a comparison of these markers can provide diagnostic information. 
     As used herein, the phrase “determining the prognosis” refers to methods by which the skilled artisan can predict the course or outcome of a condition in a subject. The term “prognosis” does not refer to the ability to predict the course or outcome of a condition with 100% accuracy, or even that a given course or outcome is predictably more or less likely to occur based on the methylation state of a biomarker (e.g., a DMR). Instead, the skilled artisan will understand that the term “prognosis” refers to an increased probability that a certain course or outcome will occur; that is, that a course or outcome is more likely to occur in a subject exhibiting a given condition, when compared to those individuals not exhibiting the condition. For example, in individuals not exhibiting the condition (e.g., having a normal methylation state of one or more DMR), the chance of a given outcome (e.g., suffering from an melanoma) may be very low. 
     In some embodiments, a statistical analysis associates a prognostic indicator with a predisposition to an adverse outcome. For example, in some embodiments, a methylation state different from that in a normal control sample obtained from a patient who does not have a cancer can signal that a subject is more likely to suffer from a cancer than subjects with a level that is more similar to the methylation state in the control sample, as determined by a level of statistical significance. Additionally, a change in methylation state from a baseline (e.g., “normal”) level can be reflective of subject prognosis, and the degree of change in methylation state can be related to the severity of adverse events. Statistical significance is often determined by comparing two or more populations and determining a confidence interval and/or a p value. See, e.g., Dowdy and Wearden, Statistics for Research, John Wiley &amp; Sons, New York, 1983, incorporated herein by reference in its entirety. Exemplary confidence intervals of the present subject matter are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% and 99.99%, while exemplary p values are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, and 0.0001. 
     In other embodiments, a threshold degree of change in the methylation state of a prognostic or diagnostic biomarker disclosed herein (e.g., a DMR) can be established, and the degree of change in the methylation state of the biamarker in a biological sample is simply compared to the threshold degree of change in the methylation state. A preferred threshold change in the methylation state for biomarkers provided herein is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 50%, about 75%, about 100%, and about 150%. In yet other embodiments, a “nomogram” can be established, by which a methylation state of a prognostic or diagnostic indicator (biomarker or combination of biomarkers) is directly related to an associated disposition towards a given outcome. The skilled artisan is acquainted with the use of such nomograms to relate two numeric values with the understanding that the uncertainty in this measurement is the same as the uncertainty in the marker concentration because individual sample measurements are referenced, not population averages. 
     In some embodiments, a control sample is analyzed concurrently with the biological sample, such that the results obtained from the biological sample can be compared to the results obtained from the control sample. Additionally, it is contemplated that standard curves can be provided, with which assay results for the biological sample may be compared. Such standard curves present methylation states of a biomarker as a function of assay units, e.g., fluorescent signal intensity, if a fluorescent label is used. Using samples taken from multiple donors, standard curves can be provided for control methylation states of the one or more biomarkers in normal tissue, as well as for “at-risk” levels of the one or more biomarkers in tissue taken from donors with metaplasia or from donors with a melanoma. In certain embodiments of the method, a subject is identified as having metaplasia upon identifying an aberrant methylation state of one or more DMR provided herein in a biological sample obtained from the subject. In other embodiments of the method, the detection of an aberrant methylation state of one or more of such biomarkers in a biological sample obtained from the subject results in the subject being identified as having cancer. 
     The analysis of markers can be carried out separately or simultaneously with additional markers within one test sample. For example, several markers can be combined into one test for efficient processing of a multiple of samples and for potentially providing greater diagnostic and/or prognostic accuracy. In addition, one skilled in the art would recognize the value of testing multiple samples (for example, at successive time points) from the same subject. Such testing of serial samples can allow the identification of changes in marker methylation states over time. Changes in methylation state, as well as the absence of change in methylation state, can provide useful information about the disease status that includes, but is not limited to, identifying the approximate time from onset of the event, the presence and amount of salvageable tissue, the appropriateness of drug therapies, the effectiveness of various therapies, and identification of the subject&#39;s outcome, including risk of future events. 
     The analysis of biomarkers can be carried out in a variety of physical formats. For example, the use of microtiter plates or automation can be used to facilitate the processing of large numbers of test samples. Alternatively, single sample formats could be developed to facilitate immediate treatment and diagnosis in a timely fashion, for example, in ambulatory transport or emergency room settings. 
     In some embodiments, the subject is diagnosed as having melanoma if, when compared to a control methylation state, there is a measurable difference in the methylation state of at least one biomarker in the sample. Conversely, when no change in methylation state is identified in the biological sample, the subject can be identified as not having melanoma, not being at risk for the cancer, or as having a low risk of the cancer. In this regard, subjects having the cancer or risk thereof can be differentiated from subjects having low to substantially no cancer or risk thereof. Those subjects having a risk of developing melanoma can be placed on a more intensive and/or regular screening schedule, including endoscopic surveillance. On the other hand, those subjects having low to substantially no risk may avoid being subjected to additional testing for melanoma (e.g., invasive procedure), until such time as a future screening, for example, a screening conducted in accordance with the present technology, indicates that a risk of melanoma has appeared in those subjects. 
     As mentioned above, depending on the embodiment of the method of the present technology, detecting a change in methylation state of the one or more biomarkers can be a qualitative determination or it can be a quantitative determination. As such, the step of diagnosing a subject as having, or at risk of developing, melanoma indicates that certain threshold measurements are made, e.g., the methylation state of the one or more biomarkers in the biological sample varies from a predetermined control methylation state. In some embodiments of the method, the control methylation state is any detectable methylation state of the biomarker. In other embodiments of the method where a control sample is tested concurrently with the biological sample, the predetermined methylation state is the methylation state in the control sample. In other embodiments of the method, the predetermined methylation state is based upon and/or identified by a standard curve. In other embodiments of the method, the predetermined methylation state is a specifically state or range of state. As such, the predetermined methylation state can be chosen, within acceptable limits that will be apparent to those skilled in the art, based in part on the embodiment of the method being practiced and the desired specificity, etc. 
     Further with respect to diagnostic methods, a preferred subject is a vertebrate subject. A preferred vertebrate is warm-blooded; a preferred warm-blooded vertebrate is a mammal. A preferred mammal is most preferably a human. As used herein, the term “subject’ includes both human and animal subjects. Thus, veterinary therapeutic uses are provided herein. As such, the present technology provides for the diagnosis of mammals such as humans, as well as those mammals of importance due to being endangered, such as Siberian tigers; of economic importance, such as animals raised on farms for consumption by humans; and/or animals of social importance to humans, such as animals kept as pets or in zoos. Examples of such animals include but are not limited to: carnivores such as cats and dogs; swine, including pigs, hogs, and wild boars; ruminants and/or ungulates such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels; and horses. Thus, also provided is the diagnosis and treatment of livestock, including, but not limited to, domesticated swine, ruminants, ungulates, horses (including race horses), and the like. 
     The presently-disclosed subject matter further includes a system for diagnosing melanoma and/or a specific form of melanoma (e.g., metastatic melanoma, primary cutaneous melanoma) in a subject. The system can be provided, for example, as a commercial kit that can be used to screen for a risk of melanoma or diagnose melanoma in a subject from whom a biological sample has been collected. An exemplary system provided in accordance with the present technology includes assessing the methylation state of a DMR as provided in Tables 1A, 2A, 7A, 8A, and 9. 
     EXAMPLES 
     Example I 
     Materials and Methods 
     Tissue and blood were obtained from Mayo Clinic biospecimen repositories with institutional IRB oversight. Samples were chosen with strict adherence to subject research authorization and inclusion/exclusion criteria. Cancers consisted of 21 metastatic melanomas. Controls included 15 non-neoplastic skin epidermis samples, 16 benign melanocytic nevi, and 36 whole blood derived leukocytes. Tissues were macro-dissected and histology reviewed by an expert pathologist. Samples were age matched, randomized, and blinded. DNA was purified using the QIAamp DNA Tissue Mini kit and QIAamp DNA Blood Mini kit (Qiagen, Valencia Calif.), respectively. DNA was re-purified with AMPure XP beads (Beckman-Coulter, Brea Calif.) and quantified by PicoGreen (Thermo-Fisher, Waltham Mass.). DNA integrity was assessed using qPCR. 
     RRBS sequencing libraries were prepared following the Meissner protocol (see, Gu et al. Nature Protocols 2011 April; 6(4):468-81) with modifications. Samples were combined in a 4-plex format and sequenced by the Mayo Genomics Facility on the Illumina HiSeq 2500 instrument (Illumina, San Diego Calif.). Reads were processed by Illumina pipeline modules for image analysis and base calling. Secondary analysis was performed using SAAP-RRBS, a Mayo developed bioinformatics suite. Briefly, reads were cleaned-up using Trim-Galore and aligned to the GRCh37/hgl9 reference genome build with BSMAP. Methylation ratios were determined by calculating C/(C+T) or conversely, G/(G+A) for reads mapping to reverse strand, for CpGs with coverage ≥10× and base quality score ≥20. 
     Individual CpGs were ranked by hypermethylation ratio, namely the number of methylated cytosines at a given locus over the total cytosine count at that site. For cases, the ratios were required to be ≥0.20 (20%); for tissue controls, ≤0.05 (5%) tissue vs tissue analysis; ≥0.20 (20%) tissue vs buffy coat; for buffy coat controls, ≤0.01 (1%). CpGs which did not meet these criteria were discarded. Subsequently, candidate CpGs were binned by genomic location into DMRs (differentially methylated regions) ranging from approximately 40-220 bp with a minimum cut-off of 5 CpGs per region. DMRs with excessively high CpG density (&gt;30%) were excluded to avoid GC-related amplification problems in the validation phase. For each candidate region, a 2-D matrix was created which compared individual CpGs in a sample-to-sample fashion for both cases and controls. These CpG matrices were then compared back to the reference sequence to assess whether genomically contiguous methylation sites had been discarded during the initial filtering. From this subset of regions, final selections required coordinated and contiguous hypermethylation (in cases) of individual CpGs across the DMR sequence on a per sample level. Conversely, control samples had to have at least 10-fold less methylation than cases and the CpG pattern had to be more random and less coordinated. At least 10% of cancer samples were required to have at least a 50% hypermethylation ratio for every CpG site within the DMR. 
     In a separate analysis, a proprietary DMR identification pipeline and regression package was utilized to derive DMRs based on average methylation values of the CpG. The difference in average methylation percentage was compared between malignant melanoma cases, tissue controls and buffy coat controls; a tiled reading frame within 100 base pairs of each mapped CpG was used to identify DMRs where control methylation was &lt;5%; DMRs were only analyzed if the total depth of coverage was 10 reads per subject on average and the variance across subgroups was &gt;0. Assuming a biologically relevant increase in the odds ratio of &gt;3× and a coverage depth of 10 reads, ≥18 samples per group were required to achieve 80% power with a two-sided test at a significance level of 5% and assuming binomial variance inflation factor of 1. 
     Following regression, DMRs were ranked by p-value, area under the receiver operating characteristic curve (AUC) and fold-change difference between cases and all controls. No adjustments for false discovery were made during this phase as independent validation was planned a priori. 
     A subset of the DMRs was chosen for further development. The criteria were primarily the logistic-derived area under the ROC curve metric which provides a performance assessment of the discriminant potential of the region. An AUC of 0.85 was chosen as the cut-off. In addition, the methylation fold-change ratio (average cancer hypermethylation ratio/average control hypermethylation ratio) was calculated and a lower limit of 10 was employed for tissue vs tissue comparisons and 20 for the tissue vs buffy coat comparisons. P values were required to be less than 0.01. DMRs had to be listed in both the average and individual CpG selection processes. Quantitative methylation specific PCR (qMSP) primers were designed for candidate regions using MethPrimer (Li LC and Dahiya R. Bioinformatics 2002 November; 18(11):1427-31) and QC checked on 20 ng (6250 equivalents) of positive and negative genomic methylation controls. Multiple annealing temperatures were tested for optimal discrimination. Validation was performed in two stages of qMSP. The first consisted of re-testing the sequenced DNA samples. This was done to verify that the DMRs were truly discriminant and not the result of over-fitting the extremely large next generation sequencing (NGS) datasets. The second utilized a larger set of independent samples: metastatic melanoma from 35 patients, primary melanoma from 26 patients, and 73 control samples (47 benign precursor lesions or normal skin, and 26 healthy buffy coat samples. 
     Tissues were identified as before, with expert clinical and pathological review. DNA purification was performed using the Qiagen QIAmp FFPE tissue kit. The EZ-96 DNA Methylation kit (Zymo Research, Irvine Calif.) was used for the bisulfite conversion step. 10 ng of converted DNA (per marker) was amplified using SYBR Green detection on Roche 480 LightCyclers (Roche, Basel Switzerland). Serially diluted universal methylated genomic DNA (Zymo Research) was used as a quantitation standard. A CpG agnostic ACTB (0-actin) assay was used as an input reference and normalization control. Results were expressed as methylated copies (specific marker)/copies of ACTB. 
     Results were analyzed logistically for individual MDMs (methylated DNA marker) performance. For combinations of markers, two techniques were used. First, the rPart technique was applied to the entire MDM set and limited to combinations of 3 MDMs, upon which an rPart predicted probability of cancer was calculated. The second approach used random forest regression (rForest) which generated 500 individual rPart models that were fit to boot strap samples of the original data (roughly ⅔ of the data for training) and used to estimate the cross-validation error (⅓ of the data for testing) of the entire MDM panel and was repeated 500 times. to avoid spurious splits that either under- or overestimate the true cross-validation metrics. Results were then averaged across the 500 iterations. 
     Results 
     A proprietary methodology of sample preparation, sequencing, analyses, and filters was utilized to identify and narrow differentially methylated regions (DMRs) to those which would pinpoint skin cancers and excel in a clinical testing environment. From the tissue-to-tissue analysis, 124 hypermethylated malignant melanomas (MM) DMRs were identified (Tables TA and 1B). They included MM specific regions as well as those regions that targeted a more universal cancer spectrum. The tissue to leukocyte (buffy coat) analysis yielded 38 hypermethylated MM+epidermis tissue DMRs with less than 1% noise in WBCs (Tables 2A and 2B). 
     From the tissue and buffy marker groups, 40 candidate markers were chosen for an initial pilot. Methylation-specific PCR assays were developed and tested on two rounds of tissue samples; those that were sequenced (frozen) and larger independent cohorts (FFPE). Short amplicon primers (&lt;150 bp) were designed to target the most discriminant CpGs within a DMR and tested on controls to ensure that fully methylated fragments amplified robustly and in a linear fashion; that unmethylated and/or unconverted fragments did not amplify. The 80 primer sequences are listed in Table 3. 
     The results from stage one validation were analyzed logistically to determine AUC and fold change. The analyses for the tissue and buffy coat controls were run separately. Results are highlighted in Table 4. The degree of red shading indicates the discrimination strength of the marker assay. A number of assays were 100% discriminant in MM from buffy coat samples and one was 100% in the MM vs control tissue analysis. 
     These results provided a rich source of highly performing candidates to take into independent sample testing. Of the original 40 assays, 32 were selected. Most fell within the AUC range of 0.90-1.00, but others were included which had extremely high fold change (FC) numbers (very little background) and/or those which exhibited complementarity with other methylated DNA markers (MDMS). All assays demonstrated high analytical performance—linearity, efficiency, sequence specificity (assessed using melt curve analysis), and strong amplification. 
     In round 2 validation, as in the previous step, the entire sample and marker set was run in one batch. ˜10 ng of FFPE-derived sample DNA was run per marker—350 total. MM vs normal tissue and buffy coat results for individual MDMs are listed in Table 5A. On receiver operator characteristics analyses of individual marker candidates, areas under the curve (AUCs) for the MM vs control tissue comparison ranged from 0.43 to 0.97 (Table 5B); median AUC was 0.835. At 100% specificity, a cross-validated panel of 5 MDMs (MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763 (using primer SEQ ID Nos. 47 and 48), MAX.chr7.155259597-155259763 (using primer SEQ ID Nos. 49 and 50), FOXL2NB, and HOXA9_A) yielded a sensitivity of 33/35 cases (94.3% (95% CI, 86.6-100%)) for metastatic melanoma and 22/26 cases (84.6% (95% CI, 70.7-98.5%)) for primary melanoma (Table 5C). For the MM vs buffy coat comparison, AUCs ranged from 0.80 to 0.98 and methylation fold change ratios were &gt;20 with a median of 64 (Table 6). 
     Whole methylome sequencing, stringent filtering criteria, and biological validation yielded outstanding candidate MDMs for malignant melanoma. A panel of five novel MDMs (MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763 (using primer SEQ ID Nos. 47 and 48), MAX.chr7.155259597-155259763 (using primer SEQ ID Nos. 49 and 50), FOXL2NB, and HOXA9_A) assayed on tissue and undetectable in normal buffy coat achieves very high discrimination between melanoma and benign control tissues. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 1A 
               
               
                   
               
               
                   
                   
                   
                 DMR Start-End Positions 
               
               
                 DMR No. 
                 Gene Annotation 
                 Chromosome No. 
                 (GRCh37/hg19) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 ACTR3C 
                 7 
                 150020206-150020574 
               
               
                 2 
                 ADAM8 
                 10 
                 135090076-135090566 
               
               
                 3 
                 ALOX12B 
                 17 
                 7976119-7976483 
               
               
                 4 
                 ALX3 
                 1 
                 110612988-110613158 
               
               
                 5 
                 ANKRD33B 
                 5 
                 10565042-10565297 
               
               
                 6 
                 ASB2 
                 14 
                 94405842-94406155 
               
               
                 7 
                 ATP8B1 
                 18 
                 55469253-55469316 
               
               
                 8 
                 BANK1 
                 4 
                 102711871-102711992 
               
               
                 9 
                 BARHL1 
                 9 
                 135462616-135462735 
               
               
                 10 
                 BOLL 
                 2 
                 198651427-198651480 
               
               
                 11 
                 BTBD19 
                 1 
                 45279368-45279564 
               
               
                 12 
                 C10orf55 
                 10 
                 75670653-75670789 
               
               
                 13 
                 C14orf50 
                 14 
                 65016722-65016815 
               
               
                 14 
                 C1QL3 
                 10 
                 16562465-16562672 
               
               
                 15 
                 C2orf82 
                 2 
                 233740970-233741417 
               
               
                 16 
                 FOXL2NB 
                 3 
                 138663981-138664076 
               
               
                 17 
                 C6orf132 
                 6 
                 42109906-42110586 
               
               
                 18 
                 CCDC109B 
                 4 
                 110480758-110481153 
               
               
                 19 
                 CD8B 
                 2 
                 87089115-87089153 
               
               
                 20 
                 CLDN10 
                 13 
                 96204805-96204874 
               
               
                 21 
                 CLIC5 
                 6 
                 45982945-45983289 
               
               
                 22 
                 CRHBP_A 
                 5 
                 76249301-76249698 
               
               
                 23 
                 CRHBP_B 
                 5 
                 76249969-76250202 
               
               
                 24 
                 CYP26A1 
                 10 
                 94834101-94834349 
               
               
                 25 
                 DAB1 
                 1 
                 58715853-58716179 
               
               
                 26 
                 DMRT3 
                 9 
                 977078-977505 
               
               
                 27 
                 F2RL1 
                 5 
                 76115146-76115392 
               
               
                 28 
                 FAM174B_A 
                 15 
                 93198406-93198607 
               
               
                 29 
                 FAM174B_B 
                 15 
                 93199001-93199143 
               
               
                 30 
                 FGFR1 
                 8 
                 38323133-38323199 
               
               
                 31 
                 FLJ41350 
                 10 
                 102986651-102986744 
               
               
                 32 
                 FOXP1 
                 3 
                 71630795-71630964 
               
               
                 33 
                 GATA6 
                 18 
                 19745472-19745506 
               
               
                 34 
                 GFRA2 
                 8 
                 21647026-21647253 
               
               
                 35 
                 GIPC2 
                 1 
                 78511659-78511979 
               
               
                 36 
                 GJA1 
                 6 
                 121758272-121758386 
               
               
                 37 
                 GNA14 
                 9 
                 80263496-80263854 
               
               
                 38 
                 GREM1 
                 15 
                 33010893-33011064 
               
               
                 39 
                 GSN 
                 9 
                 124062312-124062460 
               
               
                 40 
                 GSTO2 
                 10 
                 106034646-106034802 
               
               
                 41 
                 HEYL 
                 1 
                 40105255-40105487 
               
               
                 42 
                 HIST1H3G 
                 6 
                 26273747-26273884 
               
               
                 43 
                 HLA-J 
                 6 
                 29974345-29974746 
               
               
                 44 
                 HLF 
                 17 
                 53343188-53343401 
               
               
                 45 
                 HOPX 
                 4 
                 57522436-57522653 
               
               
                 46 
                 HOXA9_A 
                 7 
                 27209628-27209739 
               
               
                 47 
                 HOXB5_A 
                 17 
                 46674939-46675240 
               
               
                 48 
                 IGFBP5 
                 2 
                 217559020-217559578 
               
               
                 49 
                 KCNQ4_A 
                 1 
                 41284410-41284590 
               
               
                 50 
                 LAMA3 
                 18 
                 21269793-21270254 
               
               
                 51 
                 LBX2_A 
                 2 
                 74725933-74726331 
               
               
                 52 
                 LBX2_B 
                 2 
                 74726449-74726634 
               
               
                 53 
                 LOC100128977 
                 17 
                 43974551-43974611 
               
               
                 54 
                 LOC648809_A 
                 15 
                 84749077-84749238 
               
               
                 55 
                 MAL_A 
                 2 
                 95692316-95692525 
               
               
                 56 
                 MAL_B 
                 2 
                 95691099-95691322 
               
               
                 57 
                 MAX.chr1.208132378-208132686 
                 1 
                 208132378-208132686 
               
               
                 58 
                 MAX.chr1.212838668-212838781 
                 1 
                 212838668-212838781 
               
               
                 59 
                 MAX.chr1.29101688-29101792 
                 1 
                 29101688-29101792 
               
               
                 60 
                 MAX.chr1.32238359-32238526 
                 1 
                 32238359-32238526 
               
               
                 61 
                 MAX.chr10.22541869-22541953 
                 10 
                 22541869-22541953 
               
               
                 62 
                 MAX.chr10.22624234-22624571 
                 10 
                 22624234-22624571 
               
               
                 63 
                 MAX.chr10.29011068-29011271 
                 10 
                 29011068-29011271 
               
               
                 64 
                 MAX.chr10.62492690-62492812 
                 10 
                 62492690-62492812 
               
               
                 65 
                 MAX.chr11.14926602-14927148 
                 11 
                 14926602-14927148 
               
               
                 66 
                 MAX.chr13.29106812-29106917 
                 13 
                 29106812-29106917 
               
               
                 67 
                 MAX.chr13.33924444-33924575 
                 13 
                 33924444-33924575 
               
               
                 68 
                 MAX.chr15.53097592-53097737 
                 15 
                 53097592-53097737 
               
               
                 69 
                 MAX.chr15.53098014-53098109 
                 15 
                 53098014-53098109 
               
               
                 70 
                 MAX.chr16.21675437-21675543 
                 16 
                 21675437-21675543 
               
               
                 71 
                 MAX.chr17.29335857-29336076 
                 17 
                 29335857-29336076 
               
               
                 72 
                 MAX.chr17.6559968-6560221 
                 17 
                 6559968-6560221 
               
               
                 73 
                 MAX.chr17.73073682-73073830 
                 17 
                 73073682-73073830 
               
               
                 74 
                 MAX.chr2.162283698-162283742 
                 2 
                 162283698-162283742 
               
               
                 75 
                 MAX.chr2.223170699-223170942 
                 2 
                 223170699-223170942 
               
               
                 76 
                 MAX.chr2.233285553-233285863 
                 2 
                 233285553-233285863 
               
               
                 77 
                 MAX.chr2.237082545-237082555 
                 2 
                 237082545-237082555 
               
               
                 78 
                 MAX.chr2.97193120-97193287 
                 2 
                 97193120-97193287 
               
               
                 79 
                 MAX.chr2.97193478-97193562 
                 2 
                 97193478-97193562 
               
               
                 80 
                 MAX.chr20.21491437-21491503 
                 20 
                 21491437-21491503 
               
               
                 81 
                 MAX.chr20.3229151-3229791 
                 20 
                 3229151-3229791 
               
               
                 82 
                 MAX.chr4.4867535-4867655 
                 4 
                 4867535-4867655 
               
               
                 83 
                 MAX.chr5.139144115-139144199 
                 5 
                 139144115-139144199 
               
               
                 84 
                 MAX.chr5.42952363-42952548 
                 5 
                 42952363-42952548 
               
               
                 85 
                 MAX.chr5.42992655-42992768 
                 5 
                 42992655-42992768 
               
               
                 86 
                 MAX.chr5.60921627-60921853 
                 5 
                 60921627-60921853 
               
               
                 87 
                 MAX.chr5.76476081-76476350 
                 5 
                 76476081-76476350 
               
               
                 88 
                 MAX.chr5.77268600-77268725 
                 5 
                 77268600-77268725 
               
               
                 89 
                 MAX.chr6.157557371-157557657 
                 6 
                 157557371-157557657 
               
               
                 90 
                 MAX.chr6.26234019-26234258 
                 6 
                 26234019-26234258 
               
               
                 91 
                 MAX.chr6.29521537-29521696 
                 6 
                 29521537-29521696 
               
               
                 92 
                 MAX.chr6.45631468-45631500 
                 6 
                 45631468-45631500 
               
               
                 93 
                 MAX.chr6.99295949-99295996 
                 6 
                 99295949-99295996 
               
               
                 94 
                 MAX.chr7.149120005-149120361 
                 7 
                 149120005-149120361 
               
               
                 95 
                 MAX.chr7.156409579-156409711 
                 7 
                 156409579-156409711 
               
               
                 96 
                 MAX.chr8.82543201-82543257 
                 8 
                 82543201-82543257 
               
               
                 97 
                 ME3 
                 11 
                 86382754-86383237 
               
               
                 98 
                 MGA 
                 15 
                 41952429-41953195 
               
               
                 99 
                 MIR155HG 
                 21 
                 26934273-26934633 
               
               
                 100 
                 MYO5A 
                 15 
                 52821789-52821964 
               
               
                 101 
                 NPR3 
                 5 
                 32713578-32713695 
               
               
                 102 
                 NRARP 
                 9 
                 140200138-140200258 
               
               
                 103 
                 OLIG2 
                 21 
                 34395395-34395485 
               
               
                 104 
                 OSR2 
                 8 
                 99952116-99952873 
               
               
                 105 
                 OXT 
                 20 
                 3052495-3052618 
               
               
                 106 
                 OXTR 
                 3 
                 8809811-8809899 
               
               
                 107 
                 PLEKHA7 
                 11 
                 17035319-17035436 
               
               
                 108 
                 PROM1_A 
                 4 
                 16084793-16085386 
               
               
                 109 
                 RGMA 
                 15 
                 93632651-93632755 
               
               
                 110 
                 RNF220_A 
                 1 
                 44883618-44883741 
               
               
                 111 
                 SIX4_A 
                 14 
                 61188480-61188614 
               
               
                 112 
                 SIX4_B 
                 14 
                 61188901-61189191 
               
               
                 113 
                 SLC5A2 
                 16 
                 31498792-31499039 
               
               
                 114 
                 STAT4 
                 2 
                 192015272-192015447 
               
               
                 115 
                 SULT1A1 
                 16 
                 28634532-28634986 
               
               
                 116 
                 SYNPO 
                 5 
                 150004289-150004715 
               
               
                 117 
                 TAL1 
                 1 
                 47698036-47698142 
               
               
                 118 
                 TLX1NB 
                 10 
                 102881103-102881247 
               
               
                 119 
                 TMEM30B 
                 14 
                 61747319-61748246 
               
               
                 120 
                 TNFRSF10C 
                 8 
                 22960641-22960743 
               
               
                 121 
                 TNRC18 
                 7 
                 5467547-5467777 
               
               
                 122 
                 TSPAN33 
                 7 
                 128809044-128809129 
               
               
                 123 
                 VIPR2 
                 7 
                 158938034-158938137 
               
               
                 124 
                 ZIC1 
                 3 
                 147130252-147130259 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 1B 
               
               
                   
               
               
                   
                   
                 Area Under 
                   
                   
               
               
                 DMR No. 
                 Gene Annotation 
                 Curve 
                 Fold-Change 
                 p-value 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 ACTR3C 
                 0.89 
                 23.83 
                 0.001078 
               
               
                 2 
                 ADAM8 
                 0.8867 
                 51.35 
                 0.006575 
               
               
                 3 
                 ALOX12B 
                 0.95 
                 21.28 
                 8.69E−05 
               
               
                 4 
                 ALX3 
                 0.9368 
                 20.17 
                 2.50E−06 
               
               
                 5 
                 ANKRD33B 
                 0.8558 
                 56.29 
                 0.002555 
               
               
                 6 
                 ASB2 
                 0.8667 
                 18.82 
                 0.000224 
               
               
                 7 
                 ATP8B1 
                 0.956 
                 22.36 
                 0.009388 
               
               
                 8 
                 BANK1 
                 0.8474 
                 14.09 
                 7.01E−05 
               
               
                 9 
                 BARHL1 
                 0.8944 
                 21.63 
                 5.90E−06 
               
               
                 10 
                 BOLL 
                 0.8995 
                 4.667 
                 0.002879 
               
               
                 11 
                 BTBD19 
                 0.9283 
                 16.44 
                 1.08E−05 
               
               
                 12 
                 C10orf55 
                 0.9561 
                 68.02 
                 0.003481 
               
               
                 13 
                 C14orf50 
                 0.875 
                 145.4 
                 0.003345 
               
               
                 14 
                 C1QL3 
                 0.8481 
                 19.95 
                 1.01E−05 
               
               
                 15 
                 C2orf82 
                 0.9333 
                 157.4 
                 0.001918 
               
               
                 16 
                 FOXL2NB 
                 0.98 
                 17.62 
                   2E−06 
               
               
                 17 
                 C6orf132 
                 0.9016 
                 37.73 
                 0.004609 
               
               
                 18 
                 CCDC109B 
                 0.8467 
                 35.52 
                 0.003198 
               
               
                 19 
                 CD8B 
                 0.8462 
                 31.39 
                 0.002657 
               
               
                 20 
                 CLDN10 
                 0.9364 
                 16.41 
                 0.004922 
               
               
                 21 
                 CLIC5 
                 0.9 
                 34.97 
                 0.008759 
               
               
                 22 
                 CRHBP_A 
                 0.8833 
                 9.946 
                 0.000229 
               
               
                 23 
                 CRHBP_B 
                 0.9033 
                 10.97 
                 0.00095  
               
               
                 24 
                 CYP26A1 
                 0.9298 
                 18.73 
                 0.000592 
               
               
                 25 
                 DAB1 
                 0.9433 
                 33.88 
                 9.19E−06 
               
               
                 26 
                 DMRT3 
                 0.8632 
                 14.94 
                 0.000788 
               
               
                 27 
                 F2RL1 
                 0.9397 
                 36.65 
                 0.002797 
               
               
                 28 
                 FAM174B_A 
                 0.9762 
                 62.26 
                 1.70E−05 
               
               
                 29 
                 FAM174B_B 
                 0.9463 
                 256.7 
                 0.000721 
               
               
                 30 
                 FGFR1 
                 0.8778 
                 15.68 
                 0.000343 
               
               
                 31 
                 FLJ41350 
                 0.913 
                 43.78 
                 0.002036 
               
               
                 32 
                 FOXP1 
                 0.9333 
                 101.5 
                 0.007177 
               
               
                 33 
                 GATA6 
                 0.9515 
                 22.69 
                 0.000455 
               
               
                 34 
                 GFRA2 
                 0.9263 
                 37.03 
                 0.004077 
               
               
                 35 
                 GIPC2 
                 0.9183 
                 12.7 
                 3.40E−05 
               
               
                 36 
                 GJA1 
                 0.86 
                 58.5 
                 0.00201  
               
               
                 37 
                 GNA14 
                 0.9296 
                 33.88 
                 0.000511 
               
               
                 38 
                 GREM1 
                 0.9373 
                 30.76 
                 0.000847 
               
               
                 39 
                 GSN 
                 0.9587 
                 100.8 
                 8.70E−05 
               
               
                 40 
                 GSTO2 
                 0.94 
                 11.91 
                 0.000835 
               
               
                 41 
                 HEYL 
                 0.8737 
                 27.66 
                 0.000189 
               
               
                 42 
                 HIST1H3G 
                 0.9526 
                 58.48 
                 0.003287 
               
               
                 43 
                 HLA-J 
                 0.8783 
                 47.28 
                 0.004566 
               
               
                 44 
                 HLF 
                 0.8533 
                 47.04 
                 0.002383 
               
               
                 45 
                 HOPX 
                 0.907 
                 32.71 
                 0.002557 
               
               
                 46 
                 HOXA9_A 
                 0.9667 
                 26.5 
                 0.000298 
               
               
                 47 
                 HOXB5_A 
                 0.8667 
                 39.78 
                 0.008676 
               
               
                 48 
                 IGFBP5 
                 0.8667 
                 32.37 
                 0.002762 
               
               
                 49 
                 KCNQ4_A 
                 0.9361 
                 19.46 
                 9.99E−05 
               
               
                 50 
                 LAMA3 
                 0.9167 
                 20.02 
                 0.000165 
               
               
                 51 
                 LBX2_A 
                 0.89 
                 49.07 
                 0.003008 
               
               
                 52 
                 LBX2_B 
                 0.8767 
                 29.9 
                 0.000206 
               
               
                 53 
                 LOC100128977 
                 0.8533 
                 20.47 
                 7.32E−05 
               
               
                 54 
                 LOC648809_A 
                 0.8556 
                 22.83 
                 0.008257 
               
               
                 55 
                 MAL_A 
                 0.9228 
                 19.76 
                 0.005503 
               
               
                 56 
                 MAL_B 
                 0.869 
                 15.87 
                 0.005458 
               
               
                 57 
                 MAX.chr1.208132378-208132686 
                 0.9365 
                 38.3 
                 4.73E−06 
               
               
                 58 
                 MAX.chr1.212838668-212838781 
                 0.8609 
                 17.15 
                 0.0008  
               
               
                 59 
                 MAX.chr1.29101688-29101792 
                 0.891 
                 76.06 
                 0.004054 
               
               
                 60 
                 MAX.chr1.32238359-32238526 
                 0.9228 
                 25.66 
                 9.17E−05 
               
               
                 61 
                 MAX.chr10.22541869-22541953 
                 0.9246 
                 20.45 
                 1.47E−05 
               
               
                 62 
                 MAX.chr10.22624234-22624571 
                 0.9317 
                 54.86 
                 0.000785 
               
               
                 63 
                 MAX.chr10.29011068-29011271 
                 0.9082 
                 52.59 
                 0.003768 
               
               
                 64 
                 MAX.chr10.62492690-62492812 
                 1 
                 77.97 
                 0.000239 
               
               
                 65 
                 MAX.chr11.14926602-14927148 
                 0.9967 
                 40.58 
                 1.49E−07 
               
               
                 66 
                 MAX.chr13.29106812-29106917 
                 0.9 
                 34.6 
                 0.001974 
               
               
                 67 
                 MAX.chr13.33924444-33924575 
                 0.9533 
                 89.4 
                 0.00172  
               
               
                 68 
                 MAX.chr15.53097592-53097737 
                 0.8632 
                 48.66 
                 5.53E−06 
               
               
                 69 
                 MAX.chr15.53098014-53098109 
                 0.9544 
                 66.75 
                 3.08E−06 
               
               
                 70 
                 MAX.chr16.21675437-21675543 
                 0.8988 
                 60.5 
                 0.001252 
               
               
                 71 
                 MAX.chr17.29335857-29336076 
                 0.9333 
                 24.19 
                 0.000405 
               
               
                 72 
                 MAX.chr17.6559968-6560221 
                 0.9396 
                 62.27 
                 0.004068 
               
               
                 73 
                 MAX.chr17.73073682-73073830 
                 0.955 
                 36.76 
                 2.19E−06 
               
               
                 74 
                 MAX.chr2.162283698-162283742 
                 0.9363 
                 19.6 
                 0.000364 
               
               
                 75 
                 MAX.chr2.223170699-223170942 
                 0.8967 
                 86.51 
                 0.00616  
               
               
                 76 
                 MAX.chr2.233285553-233285863 
                 0.8902 
                 21.11 
                 0.000109 
               
               
                 77 
                 MAX.chr2.237082545-237082555 
                 0.86 
                 12.65 
                 0.00343  
               
               
                 78 
                 MAX.chr2.97193120-97193287 
                 0.8933 
                 39.54 
                 0.000521 
               
               
                 79 
                 MAX.chr2.97193478-97193562 
                 0.8575 
                 18.58 
                 0.000535 
               
               
                 80 
                 MAX.chr20.21491437-21491503 
                 0.8846 
                 61.09 
                 0.000148 
               
               
                 81 
                 MAX.chr20.3229151-3229791 
                 0.9067 
                 24.62 
                 0.000979 
               
               
                 82 
                 MAX.chr4.4867535-4867655 
                 0.8929 
                 7.205 
                 0.00046  
               
               
                 83 
                 MAX.chr5.139144115-139144199 
                 0.8526 
                 20.95 
                 6.21E−05 
               
               
                 84 
                 MAX.chr5.42952363-42952548 
                 0.8467 
                 10.55 
                 0.00043  
               
               
                 85 
                 MAX.chr5.42992655-42992768 
                 0.9118 
                 17.28 
                 0.00023  
               
               
                 86 
                 MAX.chr5.60921627-60921853 
                 0.9433 
                 18.5 
                 2.20E−05 
               
               
                 87 
                 MAX.chr5.76476081-76476350 
                 0.8933 
                 29.09 
                 0.008773 
               
               
                 88 
                 MAX.chr5.77268600-77268725 
                 0.8526 
                 28.45 
                 8.93E−05 
               
               
                 89 
                 MAX.chr6.157557371-157557657 
                 0.87 
                 23.15 
                 0.000273 
               
               
                 90 
                 MAX.chr6.26234019-26234258 
                 0.8567 
                 36.88 
                 0.003721 
               
               
                 91 
                 MAX.chr6.29521537-29521696 
                 0.9684 
                 45.37 
                 7.01E−06 
               
               
                 92 
                 MAX.chr6.45631468-45631500 
                 0.8833 
                 15.97 
                 8.12E−06 
               
               
                 93 
                 MAX.chr6.99295949-99295996 
                 0.945 
                 12.04 
                 4.07E−08 
               
               
                 94 
                 MAX.chr7.149120005-149120361 
                 0.9333 
                 21.53 
                 1.37E−05 
               
               
                 95 
                 MAX.chr7.156409579-156409711 
                 0.8907 
                 11.48 
                 0.000132 
               
               
                 96 
                 MAX.chr8.82543201-82543257 
                 0.8947 
                 24.02 
                 1.11E−06 
               
               
                 97 
                 ME3 
                 0.9083 
                 73.59 
                 0.000717 
               
               
                 98 
                 MGA 
                 0.87 
                 19.37 
                 1.86E−05 
               
               
                 99 
                 MIR155HG 
                 0.9222 
                 112.8 
                 0.001734 
               
               
                 100 
                 MYO5A 
                 0.8867 
                 12.53 
                 0.000599 
               
               
                 101 
                 NPR3 
                 0.9263 
                 64.33 
                 0.008733 
               
               
                 102 
                 NRARP 
                 0.8947 
                 42.84 
                 0.000174 
               
               
                 103 
                 OLIG2 
                 0.9323 
                 56.78 
                 0.003995 
               
               
                 104 
                 OSR2 
                 0.8933 
                 37.47 
                 0.00343  
               
               
                 105 
                 OXT 
                 0.9333 
                 33.68 
                 0.008098 
               
               
                 106 
                 OXTR 
                 0.9431 
                 123.4 
                 0.00227  
               
               
                 107 
                 PLEKHA7 
                 0.8929 
                 44.02 
                 0.00434  
               
               
                 108 
                 PROM1_A 
                 1 
                 42.96 
                 1.89E−06 
               
               
                 109 
                 RGMA 
                 0.8833 
                 23.15 
                 0.000483 
               
               
                 110 
                 RNF220_A 
                 0.8491 
                 14.86 
                 0.000128 
               
               
                 111 
                 SIX4_A 
                 0.9433 
                 111.7 
                 0.000194 
               
               
                 112 
                 SIX4_B 
                 0.8667 
                 37.88 
                 0.001949 
               
               
                 113 
                 SLC5A2 
                 0.99 
                 19.33 
                 6.60E−06 
               
               
                 114 
                 STAT4 
                 0.855 
                 66.87 
                 0.002125 
               
               
                 115 
                 SULT1A1 
                 0.8883 
                 55.01 
                 0.003744 
               
               
                 116 
                 SYNPO 
                 0.89 
                 81.82 
                 0.003408 
               
               
                 117 
                 TAL1 
                 0.93 
                 23.27 
                 0.000142 
               
               
                 118 
                 TLX1NB 
                 0.9317 
                 51.91 
                 0.000246 
               
               
                 119 
                 TMEM30B 
                 1 
                 33.4 
                 0.000109 
               
               
                 120 
                 TNFRSF10C 
                 0.8532 
                 64.33 
                 0.002406 
               
               
                 121 
                 TNRC18 
                 0.8632 
                 90.35 
                 0.003961 
               
               
                 122 
                 TSPAN33 
                 0.9489 
                 159.8 
                 0.008218 
               
               
                 123 
                 VIPR2 
                 0.9404 
                 16.65 
                 0.00045  
               
               
                 124 
                 ZIC1 
                 0.9333 
                 24.87 
                 5.79E−05 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
             
               
                 TABLE 2A 
               
               
                   
               
               
                   
                   
                   
                 DMR Start-End Positions 
               
               
                 DMR No. 
                 Gene Annotation 
                 Chromosome No. 
                 (GRCh37/hg19) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 125 
                 ACSL5 
                 10 
                 114136053-114136132 
               
               
                 126 
                 AMN 
                 14 
                 103394740-103395037 
               
               
                 127 
                 C17orf46_A 
                 17 
                 43339287-43339498 
               
               
                 128 
                 C17orf57_A 
                 17 
                 45500997-45501102 
               
               
                 129 
                 C1orf177 
                 1 
                 55266707-55266944 
               
               
                 130 
                 C2CD4D_A 
                 1 
                 151810637-151810661 
               
               
                 131 
                 CBX4 
                 17 
                 77815562-77815704 
               
               
                 132 
                 CD14 
                 5 
                 140012023-140012386 
               
               
                 133 
                 COL2A1 
                 12 
                 48398305-48398375 
               
               
                 134 
                 ECEL1 
                 2 
                 233352602-233352702 
               
               
                 135 
                 FABP5 
                 8 
                 82192408-82192921 
               
               
                 136 
                 FLJ22536_A 
                 6 
                 21666448-21666575 
               
               
                 137 
                 FRMD4A 
                 10 
                 13933763-13933786 
               
               
                 138 
                 GALNT3_A 
                 2 
                 166650352-166650518 
               
               
                 139 
                 GPR135_A 
                 14 
                 59931111-59931142 
               
               
                 140 
                 HOXB5_B 
                 17 
                 46670918-46670995 
               
               
                 141 
                 ITPKA 
                 15 
                 41787475-41787780 
               
               
                 142 
                 JSRP1 
                 19 
                 2253201-2253376 
               
               
                 143 
                 KREMEN1_A 
                 22 
                 29467629-29467723 
               
               
                 144 
                 LMX1B 
                 9 
                 129377720-129377822 
               
               
                 145 
                 LOC648809_B 
                 15 
                 84748833-84748932 
               
               
                 146 
                 MAX.chr1.110627096-110627364 
                 1 
                 110627096-110627364 
               
               
                 147 
                 MAX.chr1.162792353-162792404 
                 1 
                 162792353-162792404 
               
               
                 148 
                 MAX.chr12.30975740-30975961 
                 12 
                 30975740-30975961 
               
               
                 149 
                 MAX.chr13.20702971-20703054 
                 13 
                 20702971-20703054 
               
               
                 150 
                 MAX.chr2.233741343-233741417 
                 2 
                 233741343-233741417 
               
               
                 151 
                 MAX.chr2.71116033-71116122 
                 2 
                 71116033-71116122 
               
               
                 152 
                 MAX.chr20.21080958-21081038 
                 20 
                 21080958-21081038 
               
               
                 153 
                 MAX.chr7.155259597-155259763 
                 7 
                 1552596597-155259763  
               
               
                 154 
                 MIR375 
                 2 
                 219866541-219866574 
               
               
                 155 
                 MPZ_A 
                 1 
                 161275561-161275653 
               
               
                 156 
                 NFATC2_A 
                 20 
                 50159073-50159168 
               
               
                 157 
                 OCA2 
                 15 
                 28339956-28340185 
               
               
                 158 
                 RNF207_A 
                 1 
                 6266128-6266210 
               
               
                 159 
                 RUNX2 
                 6 
                 45387427-45387521 
               
               
                 160 
                 SCARF2 
                 22 
                 20785289-20785580 
               
               
                 161 
                 SLC17A9 
                 20 
                 61585727-61585991 
               
               
                 162 
                 STX16 
                 20 
                 57224798-57225227 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 2B 
               
               
                   
               
               
                   
                   
                 Area Under 
                   
                   
               
               
                 DMR No. 
                 Gene Annotation 
                 Curve 
                 Fold-Change 
                 p-value 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 125 
                 ACSL5 
                 0.9637 
                 119.6 
                 0.000939 
               
               
                 126 
                 AMN 
                 1 
                 287.7 
                 0.000602 
               
               
                 127 
                 C17orf46_A 
                 1 
                 220.5 
                 7.92E−05 
               
               
                 128 
                 C17orf57_A 
                 1 
                 254.7 
                 0.000177 
               
               
                 129 
                 C1orf177 
                 0.9552 
                 86.88 
                 0.000371 
               
               
                 130 
                 C2CD4D_A 
                 1 
                 182.9 
                 3.88E−05 
               
               
                 131 
                 CBX4 
                 0.9825 
                 169.4 
                 7.25E−05 
               
               
                 132 
                 CD14 
                 0.9971 
                 179 
                 3.13E−05 
               
               
                 133 
                 COL2A1 
                 0.9624 
                 349.3 
                 0.000207 
               
               
                 134 
                 ECEL1 
                 0.954 
                 94.35 
                 0.005869 
               
               
                 135 
                 FABP5 
                 0.9778 
                 93.12 
                 0.001475 
               
               
                 136 
                 FLJ22536_A 
                 0.9597 
                 277.3 
                 2.48E−05 
               
               
                 137 
                 FRMD4A 
                 0.963 
                 237 
                 0.000311 
               
               
                 138 
                 GALNT3_A 
                 1 
                 412.1 
                 0.000257 
               
               
                 139 
                 GPR135_A 
                 1 
                 121.4 
                 0.000321 
               
               
                 140 
                 HOXB5_B 
                 1 
                 242.7 
                 8.48E−06 
               
               
                 141 
                 ITPKA 
                 0.9965 
                 122.6 
                 3.92E−06 
               
               
                 142 
                 JSRP1 
                 0.9861 
                 147.3 
                 0.000618 
               
               
                 143 
                 KREMEN1_A 
                 0.9641 
                 277.1 
                 2.47E−05 
               
               
                 144 
                 LMX1B 
                 1 
                 153.7 
                 7.63E−05 
               
               
                 145 
                 LOC648809_B 
                 1 
                 138.3 
                 0.008842 
               
               
                 146 
                 MAX.chr1.110627096-110627364 
                 0.9912 
                 268.8 
                 7.46E−05 
               
               
                 147 
                 MAX.chr1.162792353-162792404 
                 0.9667 
                 105.2 
                 0.000223 
               
               
                 148 
                 MAX.chr12.30975740-30975961 
                 0.9569 
                 191.3 
                 1.57E−05 
               
               
                 149 
                 MAX.chr13.20702971-20703054 
                 0.9542 
                 139.3 
                 0.002903 
               
               
                 150 
                 MAX.chr2.233741343-233741417 
                 0.969 
                 130.8 
                 0.000742 
               
               
                 151 
                 MAX.chr2.71116033-71116122 
                 1 
                 142.4 
                 6.71E−06 
               
               
                 152 
                 MAX.chr20.21080958-21081038 
                 1 
                 505.8 
                 0.00042  
               
               
                 153 
                 MAX.chr7.155259597-155259763 
                 1 
                 217.8 
                 5.74E−05 
               
               
                 154 
                 MIR375 
                 0.9967 
                 204.7 
                 2.68E−06 
               
               
                 155 
                 MPZ_A 
                 1 
                 475.6 
                 0.001579 
               
               
                 156 
                 NFATC2_A 
                 0.9938 
                 275.9 
                 0.002118 
               
               
                 157 
                 OCA2 
                 0.9894 
                 82.08 
                 1.18E−06 
               
               
                 158 
                 RNF207_A 
                 0.9958 
                 197.1 
                 0.000713 
               
               
                 159 
                 RUNX2 
                 0.9917 
                 197.7 
                 5.71E−05 
               
               
                 160 
                 SCARF2 
                 0.9795 
                 342.6 
                 0.009978 
               
               
                 161 
                 SLC17A9 
                 0.9622 
                 118.1 
                 0.00507  
               
               
                 162 
                 STX16 
                 1 
                 1087 
                 0.000123 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 3 
               
               
                   
               
               
                   
                   
                 Seq 
                   
                 Seq 
                   
               
               
                 DMR 
                   
                 ID 
                 Forward Primer 5′-3′ 
                 ID 
                 Reverse Primer 5′-3′ 
               
               
                 No. 
                 Gene Annotation 
                 No. 
                 Sequence 
                 No. 
                 Sequence 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 12 
                 C10orf55 
                 1 
                 TTAGGTGTATGGGAGG 
                 2 
                 AAAAAAAACGCGATCT 
               
               
                   
                   
                   
                 AAGTACGGA 
                   
                 CCAACGAAA 
               
               
                   
               
               
                 15 
                 C2orf82 
                 3 
                 CGTCGHUGHGGTT 
                 4 
                 ACCGAACGCGACCCC 
               
               
                   
                   
                   
                 ATTTGCGTG 
                   
                 TTTATTCG 
               
               
                   
               
               
                 16 
                 FOXL2NB 
                 5 
                 TATGGATTGTACGGTA 
                 6 
                 ACGCAAACCTCTTAAC 
               
               
                   
                   
                   
                 GTCGGGCGG 
                   
                 CTTCTCTCACAAATCG 
               
               
                   
               
               
                 21 
                 CLIC5 
                 7 
                 GGCGTTGGTTTGCGG 
                 8 
                 CACCCACCAAAACTCG 
               
               
                   
                   
                   
                 AAAGGTTAC 
                   
                 AAATACGCT 
               
               
                   
               
               
                 29 
                 FAM174B_B 
                 9 
                 TCGTCGTCGTTATTAT 
                 10 
                 ATAACCTACCCCGCCC 
               
               
                   
                   
                   
                 TAATACGTT 
                   
                 ACGT 
               
               
                   
               
               
                 136 
                 FLJ22536_A 
                 11 
                 GAGTTTGGTAGTGGGA 
                 12 
                 CCGAAACGAACTAAAA 
               
               
                   
                   
                   
                 GGAGATCGT 
                   
                 CTACTCGAT 
               
               
                   
               
               
                 32 
                 FOXP1 
                 13 
                 GGGGCGATTTTTAGTA 
                 14 
                 CTACCCTCTCTATCCT 
               
               
                   
                   
                   
                 AGTTTTTTTCGT 
                   
                 AACCCCTCGAC 
               
               
                   
               
               
                 138 
                 GALNT3_A 
                 15 
                 TCGGGATTTTTTTAGG 
                 16 
                 GAAAAAACGACCGCA 
               
               
                   
                   
                   
                 GGTAATCGA 
                   
                 AAAATTCACGTA 
               
               
                   
               
               
                 45 
                 HOPX 
                 17 
                 TTGTATTTTTGTTTGCG 
                 18 
                 CCGAAAATCGATAAAA 
               
               
                   
                   
                   
                 ACGGGGGC 
                   
                 ACCCGCGAA 
               
               
                   
               
               
                 46 
                 HOXA9_A 
                 19 
                 TTTTTTTTAGTTGTTCG 
                 20 
                 GACACTCCTTAAAACC 
               
               
                   
                   
                   
                 TTCGTCGG 
                   
                 ACGCGTT 
               
               
                   
               
               
                 141 
                 ITPKA 
                 21 
                 GGGTTTATAAGTTCGG 
                 22 
                 CACCCAACACCTAACG 
               
               
                   
                   
                   
                 AGGTCGA 
                   
                 ACGA 
               
               
                   
               
               
                 49 
                 KCNQ4_A 
                 23 
                 GGGCGAGGTTAGAGG 
                 24 
                 GCCTCCTACTAAAACT 
               
               
                   
                   
                   
                 GGTTGTTHC 
                   
                 CCAACCCCCG 
               
               
                   
               
               
                 144 
                 LMX1B 
                 25 
                 ACGTCGCGTATTGTAA 
                 26 
                 CTTCCTAATACGAATC 
               
               
                   
                   
                   
                 ATATTTTTCGTG 
                   
                 AACGAATCGTCC 
               
               
                   
               
               
                 146 
                 MAX.chr1.1106270 
                 27 
                 CGAAAGATCGATCGAT 
                 28 
                 TTATAACGACGAATTC 
               
               
                   
                 96-110627364 
                   
                 TGTTCGACGG 
                   
                 CGAAA 
               
               
                   
               
               
                 61 
                 MAX.chr10.225418 
                 29 
                 CGGTCGGAATTTCGGT 
                 30 
                 AACACTAACCGCGCCT 
               
               
                   
                 69-22541953 
                   
                 TTTCGC 
                   
                 AATATCGCTA 
               
               
                   
               
               
                 64 
                 MAX.chr10.624926 
                 31 
                 CGGGATAGAGGCGAG 
                 32 
                 GAATATTTCCAAACAA 
               
               
                   
                 90-62492812 
                   
                 AGTTCGAATTC 
                   
                 AAACCCCAAATCCTCG 
               
               
                   
               
               
                 66 
                 MAX.chr13.291068 
                 33 
                 CGGTTTTGATTTTAGG 
                 34 
                 TTAACTATCGAAAACG 
               
               
                   
                 12-29106917 
                   
                 GCGT 
                   
                 ATTTCCGCGAA 
               
               
                   
               
               
                 73 
                 MAX.chr17.730736 
                 35 
                 TTTTTCGAGTCGTTTTA 
                 36 
                 GAACTCCGAACGCCG 
               
               
                   
                 82-73073830 
                   
                 TTTCGCGG 
                   
                 CTTAAACGTA 
               
               
                   
               
               
                 151 
                 MAX.chr2.7111603 
                 37 
                 ATTTTTGATTATGGGTT 
                 38 
                 GAATTCACCTCGCCCC 
               
               
                   
                 3-71116122 
                   
                 TTGGTCGG 
                   
                 TCGCT 
               
               
                   
               
               
                 152 
                 MAX.chr20.210809 
                 39 
                 TAAATAAAATATTGGTT 
                 40 
                 CGAACTAACGCTAAAC 
               
               
                   
                 58-21081038 
                   
                 TGTAGACGGACGCGG 
                   
                 TCGCGCGAT 
               
               
                   
               
               
                 Me 
                 MAX.chr5.4299265 
                 41 
                 GGGGCGATAGTATTTA 
                 42 
                 TCACCCCAACTAAAAA 
               
               
                   
                 5-42992768 
                   
                 GGTCGTCGA 
                   
                 AACTCCGAA 
               
               
                   
               
               
                 86 
                 MAX.chr5.6092162 
                 43 
                 TCGTAGAGGTTATCGG 
                 44 
                 AATACCCGAACCACAA 
               
               
                   
                 7-60921853 
                   
                 ATATAGCGA 
                   
                 AACCCGCC 
               
               
                   
               
               
                 91 
                 MAX.chr6.2952153 
                 45 
                 TTGCGGAGGCGACGG 
                 46 
                 CAAAATAACCACGCGA 
               
               
                   
                 7-29521696 
                   
                 AGATATTATC 
                   
                 ACGACGAA 
               
               
                   
               
               
                 153 
                 MAX.chr7.1552595 
                 47 
                 TCGTAGAGGGCGGAT 
                 48 
                 CGACCGAAAAACAAC 
               
               
                   
                 97-155259763 (v1) 
                   
                 ATTAAATTAGCGG 
                   
                 GTTTATTCGCT 
               
               
                   
               
               
                 153 
                 MAX.chr7.1552595 
                 49 
                 TTTTCGGCGGAGTCG 
                 50 
                 GATCTCCGCTCGCCT 
               
               
                   
                 97-155259763 (v2) 
                   
                 GGATTTTTTC 
                   
                 CGACG 
               
               
                   
               
               
                 97 
                 ME3 
                 51 
                 GTTGCGGGGTATCGG 
                 52 
                 CCTACCCTCTCCCAAA 
               
               
                   
                   
                   
                 GTTTGATTC 
                   
                 AAACGCGTC 
               
               
                   
               
               
                 99 
                 MIR155HG (v1) 
                 53 
                 CGGATAGCGGAGTTTC 
                 54 
                 AAACGTCTCCTTAATT 
               
               
                   
                   
                   
                 GAGTCGTTC 
                   
                 CCCCGCGCTT 
               
               
                   
               
               
                 99 
                 MIR155HG (v2) 
                 55 
                 TAAGCGCGGGGAATTA 
                 56 
                 CGAAAACGCGAAACTA 
               
               
                   
                   
                   
                 AGGAGACGT 
                   
                 AAATCGACGTA 
               
               
                   
               
               
                 156 
                 NFATC2_A 
                 57 
                 GTTGGCGGAGGCGGT 
                 58 
                 CGACGTACGTCCCTA 
               
               
                   
                   
                   
                 TCGAG 
                   
                 CGAAA 
               
               
                   
               
               
                 157 
                 OCA2 
                 59 
                 ATTTGTTTTGTCGGGA 
                 60 
                 CAAATAACCCAAACTC 
               
               
                   
                   
                   
                 AGGGGACGG 
                   
                 CTATACGCA 
               
               
                   
               
               
                 105 
                 OXT 
                 61 
                 CGTTTGAGAATTTTAG 
                 62 
                 CGAAACAACGAAACTA 
               
               
                   
                   
                   
                 GAGTTGAGCGG 
                   
                 AAACGCGCT 
               
               
                   
               
               
                 158 
                 RNF207_A 
                 63 
                 GAGGAGAGGTAGGAG 
                 64 
                 AATAATTCCCACTCTA 
               
               
                   
                   
                   
                 AGGTTACGG 
                   
                 CGCGAA 
               
               
                   
               
               
                 159 
                 RUNX2 
                 65 
                 TCGAAAAGATAATTAA 
                 66 
                 CGAAAACCCATTTAAC 
               
               
                   
                   
                   
                 AAATCGTACGCGT 
                   
                 CAAACCGAA 
               
               
                   
               
               
                 111 
                 SIX4_A 
                 67 
                 GGTGGTTCGGCGTAAT 
                 68 
                 CGTACGCCTTCCGCA 
               
               
                   
                   
                   
                 TAAAGCGT 
                   
                 AATATACGAA 
               
               
                   
               
               
                 162 
                 STX16 
                 69 
                 TTTTACGTAGAAATAAA 
                 70 
                 AAAAAACCGAAACCCC 
               
               
                   
                   
                   
                 GGACGCGT 
                   
                 AAAAACGAC 
               
               
                   
               
               
                 117 
                 TAL1 (v1) 
                 71 
                 GTTCGTTTTCGATAAG 
                 72 
                 AATCCCCACTCCCTCC 
               
               
                   
                   
                   
                 CGTTTCGGT 
                   
                 GATA 
               
               
                   
               
               
                 117 
                 TAL1 (v2) 
                 73 
                 AAGGTATTGTCGCGG 
                 74 
                 TAAAATAAATCATTTAA 
               
               
                   
                   
                   
                 GTTCGTTCGT 
                   
                 CCCATAATAACCGAA 
               
               
                   
               
               
                 119 
                 TMEM30B (v1) 
                 75 
                 GGTTTAGGTCGATGAA 
                 76 
                 CTATCGACCAACATCG 
               
               
                   
                   
                   
                 GGTTAGGTTCGCGTA 
                   
                 CGCTACCG 
               
               
                   
               
               
                 119 
                 TMEM30B (v2) 
                 77 
                 TTAGTTTCGTTTTCGTT 
                 78 
                 AAATACGACTCCCGAT 
               
               
                   
                   
                   
                 TAGGTGCGTTG 
                   
                 AACCCGCGA 
               
               
                   
               
               
                 122 
                 TSPAN33 
                 79 
                 CGGTTGTAGGGAGGA 
                 80 
                 ACGCCAAAAAACCCAA 
               
               
                   
                   
                   
                 TTTCGAGGAAGTTC 
                   
                 CGCA 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 4 
               
               
                   
               
               
                   
                   
                 AUC Malignant 
                   
                   
                   
               
               
                   
                   
                 Melanoma Tissue 
               
               
                   
                   
                 (MM) vs Benign 
               
               
                   
                   
                 Melanocytic 
               
               
                   
                   
                 Nevi Tissue (Nevi) 
               
               
                   
                   
                 and Normal 
                 AUC MM 
                 AUC MM 
                 AUC Nevi 
               
               
                 DMR No. 
                 Gene Annotation 
                 Tissue (Normal) 
                 vs Buffy Coat 
                 vs Nevi 
                 vs Normal 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 64 
                 MAX.chr10.62492690- 
                 0.81773 
                 0.95971 
                 0.81633 
                 0.42381 
               
               
                   
                 62492812 
               
               
                 119 
                 TMEM30B (with primer 
                 0.80624 
                 0.91941 
                 0.86224 
                 0.68333 
               
               
                   
                 SEQ ID Nos. 75 and 76) 
               
               
                 119 
                 TMEM30B (with primer 
                 0.87356 
                 1 
                 0.87755 
                 0.49524 
               
               
                   
                 SEQ ID Nos: 77 and 78) 
               
               
                 46 
                 HOXA9_A 
                 0.98851 
                 1 
                 1 
                 0.90476 
               
               
                 12 
                 C10orf55 
                 0.82266 
                 0.88828 
                 0.72959 
                 0.95476 
               
               
                 73 
                 MAX.chr17.73073682- 
                 0.9376 
                 0.96703 
                 0.94558 
                 0.55238 
               
               
                   
                 73073830 
               
               
                 122 
                 TSPAN33 
                 0.91461 
                 0.91575 
                 0.91837 
                 0.35238 
               
               
                 29 
                 FAM174B_B 
                 0.88998 
                 0.90842 
                 0.89116 
                 0.52381 
               
               
                 86 
                 MAX.chr5.60921627- 
                 0.85714 
                 0.96154 
                 0.86054 
                 0.55714 
               
               
                   
                 60921853 
               
               
                 111 
                 SIX4_A 
                 0.92611 
                 1 
                 0.92177 
                 0.40952 
               
               
                 49 
                 KCNQ4_A 
                 0.84072 
                 0.95971 
                 0.88435 
                 0.58571 
               
               
                 32 
                 FOXP1 
                 0.77833 
                 0.85348 
                 0.79592 
                 0.54286 
               
               
                 105 
                 OXT 
                 0.82594 
                 0.9304 
                 0.86054 
                 0.62381 
               
               
                 15 
                 C2orf82 
                 0.88095 
                 0.94505 
                 0.87075 
                 0.61429 
               
               
                 117 
                 TAL1 (with primer SEQ 
                 0.87356 
                 0.89377 
                 0.89626 
                 0.63571 
               
               
                   
                 ID Nos: 71 and 72) 
               
               
                 117 
                 TAL1 (with primer SEQ 
                 0.82266 
                 0.91209 
                 0.86735 
                 0.64524 
               
               
                   
                 ID Nos: 73 and 74) 
               
               
                 61 
                 MAX.chr10.22541869- 
                 0.87521 
                 0.91209 
                 0.90136 
                 0.65238 
               
               
                   
                 22541953 
               
               
                 99 
                 MIR155HG (with primer 
                 0.91954 
                 0.92674 
                 0.92177 
                 0.62857 
               
               
                   
                 SEQ ID Nos: 53 and 54) 
               
               
                 99 
                 MIR155HG (with primer 
                 0.92447 
                 0.94139 
                 0.91837 
                 0.4619 
               
               
                   
                 SEQ ID Nos: 55 and 56) 
               
               
                 85 
                 MAX.chr5.42992655- 
                 0.92939 
                 0.98535 
                 0.93878 
                 0.61905 
               
               
                   
                 42992768 
               
               
                 97 
                 ME3 
                 0.84401 
                 0.92308 
                 0.85374 
                 0.38571 
               
               
                 45 
                 HOPX 
                 0.85468 
                 0.88095 
                 0.85034 
                 0.50952 
               
               
                 21 
                 CLIC5 
                 0.7619 
                 0.79121 
                 0.77211 
                 0.48095 
               
               
                 66 
                 MAX.chr13.29106812- 
                 0.78982 
                 0.8022 
                 0.80612 
                 0.44762 
               
               
                   
                 29106917 
               
               
                 91 
                 MAX.chr6.29521537- 
                 0.97865 
                 1 
                 0.97619 
                 0.53571 
               
               
                   
                 29521696 
               
               
                 16 
                 FOXL2NB 
                 0.90969 
                 1 
                 0.95238 
                 0.6619 
               
               
                 136 
                 FLJ22536_A 
                 0.95567 
                 0.99267 
                 0.95578 
                 0.64762 
               
               
                 138 
                 GALNT3_A 
                 0.82759 
                 1 
                 0.65646 
                 0.99048 
               
               
                 141 
                 ITPKA 
                 0.68309 
                 1 
                 0.59864 
                 0.69524 
               
               
                 144 
                 LMX1B 
                 0.76026 
                 0.99267 
                 0.59184 
                 0.94286 
               
               
                 146 
                 MAX.chr1.110627096- 
                 0.85057 
                 0.85714 
                 0.84354 
                 0.53571 
               
               
                   
                 110627364 
               
               
                 151 
                 MAX.chr2.71116033- 
                 0.95895 
                 1 
                 0.93878 
                 0.7619 
               
               
                   
                 71116122 
               
               
                 152 
                 MAX.chr20.21080958- 
                 0.94089 
                 0.9707 
                 0.96939 
                 0.82857 
               
               
                   
                 21081038 
               
               
                 153 
                 MAX.chr7.155259597- 
                 1 
                 1 
                 1 
                 0.46667 
               
               
                   
                 155259763 (with primer 
               
               
                   
                 SEQ ID Nos: 47 and 48) 
               
               
                 153 
                 MAX.chr7.155259597- 
                 0.99343 
                 1 
                 0.9932 
                 0.58571 
               
               
                   
                 155259763 (with primer 
               
               
                   
                 SEQ ID Nos: 49 and 50) 
               
               
                 156 
                 NFATC2_A 
                 0.74713 
                 0.97253 
                 0.71429 
                 0.7619 
               
               
                 157 
                 OCA2 
                 0.84893 
                 1 
                 0.78912 
                 0.92857 
               
               
                 158 
                 RNF207_A 
                 0.8046 
                 0.96337 
                 0.65646 
                 0.98571 
               
               
                 159 
                 RUNX2 
                 0.86371 
                 0.95604 
                 0.87075 
                 0.61429 
               
               
                 162 
                 STX16 
                 0.94253 
                 1 
                 0.88095 
                 1 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5A 
               
             
            
               
                   
               
               
                 AUC—Area under the Curve (95% CI) &amp; Median (IQR)  
               
               
                 Marker/BTACT levels in Buffy samples 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 Cancer vs. Normal/ 
                 Buffy Samples 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 DMR  
                   
                 Cancer vs. Normal 
                 Nevus 
                   
                 25th 
                 75th 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 No. 
                 MDM 
                 AUC 
                 95% CI 
                 AUC 
                 95% CI 
                 median 
                 % 
                 % 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 46 
                 HOXA9_A 
                 0.99 
                 0.97 
                 1.01 
                 0.97 
                 0.95 
                 1.09 
                 0.001 
                 0 
                 0 
               
               
                 16 
                 FOXL2NB 
                 0.97 
                 0.94 
                 1.00 
                 0.96 
                 0.93 
                 1.00 
                 0.001 
                 0 
                 9 
               
               
                 138 
                 GALNT3_A 
                 0.96 
                 0.92 
                 1.00 
                 0.75 
                 0.66 
                 0.84 
                 0.000 
                 0 
                 9 
               
               
                 152 
                 MAX.chr20.21080958- 
                 0.96 
                 0.92 
                 1.00 
                 0.96 
                 0.91 
                 1.00 
                 0.001 
                 0 
                 0 
               
               
                   
                 21081038 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 136 
                 FLJ22536_A 
                 0.95 
                 0.91 
                 0.99 
                 0.93 
                 0.87 
                 0.98 
                 0.003 
                 0 
                 0 
               
               
                 146 
                 MAX.chr1.110627096- 
                 0.95 
                 0.89 
                 1.00 
                 0.91 
                 0.85 
                 0.97 
                 0.000 
                 0 
                 0 
               
               
                   
                 110627364 (v2) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 73 
                 MAX.chr17.73073682- 
                 0.95 
                 0.91 
                 1.00 
                 0.95 
                 0.90 
                 1.00 
                 0.000 
                 0 
                 0 
               
               
                   
                 73073830 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 117 
                 TAL1 (with primer  
                 0.94 
                 0.89 
                 0.99 
                 0.93 
                 0.88 
                 0.98 
                 0.004 
                 0 
                 0.01 
               
               
                   
                 SEQ ID Nos: 73 and 74) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 151 
                 MAX.chr2.71116033- 
                 0.94 
                 0.89 
                 0.99 
                 0.90 
                 0.84 
                 0.96 
                 0.002 
                 0 
                 0.01 
               
               
                   
                 71116122 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 153 
                 MAX.chr7.155259597- 
                 0.94 
                 0.88 
                 0.99 
                 0.93 
                 0.87 
                 0.98 
                 0.000 
                 0 
                 0 
               
               
                   
                 155259763 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 156 
                 NFATC2_A 
                 0.94 
                 0.88 
                 0.99 
                 0.83 
                 0.75 
                 0.90 
                 0.000 
                 0 
                 0 
               
               
                 158 
                 RNF207_A 
                 0.94 
                 0.89 
                 0.99 
                 0.81 
                 0.73 
                 0.89 
                 0.000 
                 0 
                 0 
               
               
                 157 
                 OCA2 
                 0.93 
                 0.87 
                 0.99 
                 0.85 
                 0.78 
                 0.92 
                 0.003 
                 0 
                 0 
               
               
                 162 
                 STX16 
                 0.93 
                 0.87 
                 0.99 
                 0.84 
                 0.76 
                 0.92 
                 0.002 
                 0 
                 0 
               
               
                 86 
                 MAX.chr5.60921627- 
                 0.92 
                 0.86 
                 0.98 
                 0.89 
                 0.82 
                 0.95 
                 0.005 
                 0 
                 0.01 
               
               
                   
                 60921853 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 141 
                 ITPKA 
                 0.92 
                 0.87 
                 0.98 
                 0.81 
                 0.72 
                 0.89 
                 0.000 
                 0 
                 0 
               
               
                 144 
                 LMX1B 
                 0.91 
                 0.85 
                 0.97 
                 0.82 
                 0.74 
                 0.90 
                 0.000 
                 0 
                 0 
               
               
                 91 
                 MAX.chr6.29521537- 
                 0.90 
                 0.83 
                 0.97 
                 0.88 
                 0.81 
                 0.95 
                 0.000 
                 0 
                 0 
               
               
                   
                 29521696 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 85 
                 MAX.chr5.42992655- 
                 0.89 
                 0.82 
                 0.96 
                 0.88 
                 0.81 
                 0.95 
                 0.001 
                 0 
                 0 
               
               
                   
                 42992768 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 99 
                 MIR155HG (with primer  
                 0.87 
                 0.80 
                 0.95 
                 0.88 
                 0.82 
                 0.95 
                 0.000 
                 0 
                 0 
               
               
                   
                 SEQ ID Nos: 53 and 54) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 99 
                 MIR155HG (with primer  
                 0.87 
                 0.79 
                 0.95 
                 0.84 
                 0.76 
                 0.92 
                 0.000 
                 0 
                 0 
               
               
                   
                 SEQ ID Nos: 55 and 56) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 159 
                 RUNX2 
                 0.87 
                 0.79 
                 0.95 
                 0.79 
                 0.71 
                 0.88 
                 0.001 
                 0 
                 0 
               
               
                 64 
                 MAX.chr10.62492690- 
                 0.84 
                 0.74 
                 0.93 
                 0.77 
                 0.69 
                 0.86 
                 0.005 
                 0 
                 0.01 
               
               
                   
                 62492812 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 12 
                 C10orf55 
                 0.84 
                 0.76 
                 0.93 
                 0.77 
                 0.69 
                 0.86 
                 0.000 
                 0 
                 0 
               
               
                 29 
                 FAM174BB 
                 0.83 
                 0.75 
                 0.92 
                 0.83 
                 0.75 
                 0.91 
                 0.001 
                 0 
                 0 
               
               
                 122 
                 TSPAN33 
                 0.82 
                 0.73 
                 0.91 
                 0.80 
                 0.72 
                 0.88 
                 0.000 
                 0 
                 0 
               
               
                 15 
                 C2orf82 
                 0.79 
                 0.70 
                 0.89 
                 0.78 
                 0.69 
                 0.87 
                 0.000 
                 0 
                 0 
               
               
                 73 
                 MAX.chr17.73073682- 
                 0.78 
                 0.69 
                 0.88 
                 0.79 
                 0.70 
                 0.88 
                 0.004 
                 0 
                 0 
               
               
                   
                 73073830 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 117 
                 TAL1 (with primer  
                 0.77 
                 0.66 
                 0.87 
                 0.80 
                 0.71 
                 0.88 
                 0.001 
                 0 
                 0 
               
               
                   
                 SEQ ID Nos: 71 and 72) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 119 
                 TMEM30B (with primer  
                 0.69 
                 0.58 
                 0.81 
                 0.73 
                 0.63 
                 0.82 
                 0.002 
                 0 
                 0 
               
               
                   
                 SEQ ID Nos. 75 and 76) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 105 
                 OXT 
                 0.51 
                 0.36 
                 0.65 
                 0.51 
                 0.40 
                 0.62 
                 0.006 
                 0 
                 0.01 
               
               
                 61 
                 MAX.chr10.22541869- 
                 0.41 
                 0.29 
                 0.52 
                 0.43 
                 0.31 
                 0.54 
                 0.000 
                 0 
                 0 
               
               
                   
                 22541953 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5B 
               
             
            
               
                   
               
               
                 Random Forest Modelling--All 32 MDMs 
               
               
                 AUC = 0.98 (Error Rate 4.63%, 0/47 = 0% Normal/Nevus, 5/61 = 8.2% Cancer) 
               
               
                 Sensitivity @ Specificity (90, 95, 97.5, 99, 100) 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Specificity 
                 Normal/Nevus 
                 Sensitivity 
                 Normal 
                 Nevus 
                 Metastatic 
                 Primary 
               
               
                   
               
               
                   90% 
                 42/47 
                 58/61 = 95.08% 
                 20/20 
                 22/27 
                 35/35 
                 23/26 
               
               
                   95% 
                 44/47 
                 58/61 = 95.08% 
                 20/20 
                 24/27 
                 35/35 
                 23/26 
               
               
                 97.5% 
                 45/47 
                 58/61 = 95.08% 
                 20/20 
                 25/27 
                 35/35 
                 23/26 
               
               
                   99% 
                 46/47 
                 58/61 = 95.08% 
                 20/20 
                 26/27 
                 35/35 
                 23/26 
               
               
                 100% 
                 47/47 
                 57/61 = 93.44% 
                 20/20 
                 27/27 
                 34/35 
                 23/26 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5C 
               
               
                   
               
             
            
               
                 Random Forest Modelling--5 MDMs (MAX.chr20.21080958-21081038. 
               
               
                 MAX.chr7.155259597-155259763 (using primer SEQ ID Nos. 47 and 48), 
               
               
                 MAX.chr7.155259597-155259763 (using primer SEQ ID Nos. 49 and 50),  
               
               
                 FOXL2NB and HOXA9_A) 
               
               
                 AUC = 0.97 (Error Rate 5.56%, 1/47 = 2.1% Normal/Nevus, 5/61 = 8.2% Cancer) 
               
               
                 Sensitivity @ Specificity (90. 95, 97.5. 99, 100) 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Specificity 
                 Normal/Nevus 
                 Sensitivity 
                 Normal 
                 Nevus 
                 Metastatic 
                 Primary 
               
               
                   
               
               
                   90% 
                 42/47 
                 59/61 = 96.72% 
                 18/20 
                 24/27 
                 35/35 
                 24/26 
               
               
                   95% 
                 44/47 
                 57/61 = 93.44% 
                 19/20 
                 25/27 
                 34/35 
                 23/26 
               
               
                 97.5% 
                 45/47 
                 56/61 = 91.80% 
                 19/20 
                 26/27 
                 33/35 
                 23/26 
               
               
                   99% 
                 46/47 
                 56/61 = 91.80% 
                 20/20 
                 26/27 
                 33/35 
                 23/26 
               
               
                  100% 
                 47/47 
                 55/61 = 90.16% 
                 20/20 
                 27/27 
                 33/35 
                 22/26 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                 Normal and Nevus 
                 Primary 
                 Metastatic 
               
               
                   
                 (n = 47) 
                 (n = 26) 
                 (n = 35) 
               
               
                   
                 Median methylation 
                 Median methylation 
                 Median methylation 
               
               
                 MDMs 
                 value (IQR) 
                 value (IQR) 
                 value (IQR) 
               
               
                   
               
               
                 HOXA9_A 
                 −0.19 (−0.83, 0.56) 
                 3.08 (2.03, 3.40) 
                 4.43 (3.60, 5.53) 
               
               
                 FOXL2NB 
                    0.0 (−0.45, 0.36) 
                 3.02 (1.68, 3.56) 
                 4.32 (3.61, 5.52) 
               
               
                 chr7.155259614-81 
                   0.08 (−0.60, 0.57) 
                 2.46 (1.68, 3.06) 
                 4.26 (2.96, 4.85) 
               
               
                 chr7.155259700-746 
                  −0.1 (−0.54, 0.53) 
                 2.63 (2.22, 3.42) 
                 4.64 (3.49, 5.34) 
               
               
                 chr20.21080958-1038 
                   0.03 (−0.29, 0.36) 
                 2.75 (1.94, 3.38) 
                 3.49 (3.04, 4.56) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 6 
               
               
                   
               
               
                   
                   
                 Cancer 
                   
                 Buffy 
                   
                   
               
               
                   
                   
                 vs Buffy 
                   
                 Samples 
               
               
                 DMR No. 
                 Gene Annotation 
                 AUC 
                 FC 
                 median 
                 25 th  % 
                 75 th  % 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 12 
                 C10orf55 
                 0.87 
                 157.28 
                 0 
                 0 
                 0 
               
               
                 15 
                 C2orf82 
                 0.82 
                 40.33 
                 0 
                 0 
                 0 
               
               
                 16 
                 FOXL2NB 
                 0.97 
                 103.01 
                 0.001 
                 0 
                 0 
               
               
                 29 
                 FAM174B_B 
                 0.90 
                 103.81 
                 0.001 
                 0 
                 0 
               
               
                 136 
                 FLJ22536_A 
                 0.98 
                 47.52 
                 0.003 
                 0 
                 0 
               
               
                 138 
                 GALNT3_A 
                 0.98 
                 62.01 
                 0 
                 0 
                 0 
               
               
                 46 
                 HOXA9_A 
                 0.97 
                 71.30 
                 0.001 
                 0 
                 0 
               
               
                 141 
                 ITPKA 
                 0.96 
                 65.57 
                 0 
                 0 
                 0 
               
               
                 144 
                 LMX1B 
                 0.95 
                 89.00 
                 0 
                 0 
                 0 
               
               
                 146 
                 MAX.chr1.110627096- 
                 0.95 
                 48.81 
                 0 
                 0 
                 0 
               
               
                   
                 110627364 
               
               
                 61 
                 MAX.chr10.22541869- 
                 0.75 
                 1076.66 
                 0 
                 0 
                 0 
               
               
                   
                 22541953 
               
               
                 64 
                 MAX.chr10.62492690- 
                 0.92 
                 7.48 
                 0.005 
                 0 
                 0.01 
               
               
                   
                 62492812 
               
               
                 73 
                 MAX.chr17.73073682- 
                 0.95 
                 23.71 
                 0.004 
                 0 
                 0 
               
               
                   
                 73073830 
               
               
                 151 
                 MAX.chr2.71116033- 
                 0.97 
                 40.38 
                 0.002 
                 0 
                 0.01 
               
               
                   
                 71116122 
               
               
                 152 
                 MAX.chr20.21080958- 
                 0.98 
                 82.25 
                 0.001 
                 0 
                 0 
               
               
                   
                 21081038 
               
               
                 85 
                 MAX.chr5.42992655- 
                 0.93 
                 114.46 
                 0.001 
                 0 
                 0 
               
               
                   
                 42992768 
               
               
                 86 
                 MAX.chr5.60921627- 
                 0.93 
                 18.91 
                 0.005 
                 0 
                 0.01 
               
               
                   
                 60921853 
               
               
                 91 
                 MAX.chr6.29521537- 
                 0.87 
                 81.07 
                 0 
                 0 
                 0 
               
               
                   
                 29521696 
               
               
                 153 
                 MAX.chr7.155259597- 
                 0.95 
                 71.71 
                 0 
                 0 
                 0 
               
               
                   
                 155259763 (with primer 
               
               
                   
                 SEQ ID Nos: 47 and 48) 
               
               
                 153 
                 MAX.chr7.155259597- 
                 0.96 
                 65.69 
                 0 
                 0 
                 0 
               
               
                   
                 155259763 (with primer 
               
               
                   
                 SEQ ID Nos: 49 and 50) 
               
               
                 99 
                 MIR155HG (with primer 
                 0.91 
                 356.84 
                 0 
                 0 
                 0 
               
               
                   
                 SEQ ID Nos: 53 and 54) 
               
               
                 99 
                 MIR155HG (with primer 
                 0.89 
                 234.33 
                 0 
                 0 
                 0 
               
               
                   
                 SEQ ID Nos: 55 and 56) 
               
               
                 156 
                 NFATC2_A 
                 0.98 
                 66.97 
                 0 
                 0 
                 0 
               
               
                 157 
                 OCA2 
                 0.97 
                 41.89 
                 0.003 
                 0 
                 0 
               
               
                 105 
                 OXT 
                 0.86 
                 7.31 
                 0.006 
                 0 
                 0.01 
               
               
                 158 
                 RNF207_A 
                 0.98 
                 62.29 
                 0 
                 0 
                 0 
               
               
                 159 
                 RUNX2 
                 0.89 
                 44.93 
                 0.001 
                 0 
                 0 
               
               
                 162 
                 STX16 
                 0.97 
                 41.23 
                 0.002 
                 0 
                 0 
               
               
                 117 
                 TAL1 (with primer SEQ ID 
                 0.88 
                 19.99 
                 0.001 
                 0 
                 0 
               
               
                   
                 Nos: 71 and 72) 
               
               
                 117 
                 TAL1 (with primer SEQ ID 
                 0.95 
                 28.09 
                 0.004 
                 0 
                 0.01 
               
               
                   
                 Nos: 73 and 74) 
               
               
                 119 
                 TMEM30BV1 
                 0.80 
                 26.91 
                 0.002 
                 0 
                 0 
               
               
                 122 
                 TSPAN33 
                 0.80 
                 103.05 
                 0 
                 0 
                 0 
               
               
                   
               
            
           
         
       
     
     Example II 
     Materials and Methods 
     Whole Genome Bisulfite Sequencing (WGBS) is an NGS approach which differs from a usual RRBS method in that the entire human genome is sequenced. RRBS is an enrichment method which uses restriction endonucleases specific to CpG rich cut sites to generate fragments from transcriptional regulatory regions of the genome. These CpG islands have been shown to exhibit differential or altered methylation profiles in numerous clinical states, most notably cancer. The benefit of RRBS is that is reduces the size of the genome to 1-2% of the total 3.2 billion nucleotides, substantially reducing cost while capturing the majority of promoters and CpG island. The downside is that is does miss a potentially substantial number of regulatory regions which either are CpG rich but do not contain the endonuclease binding sequence or those that are not part of a CpG island. Additionally, the results are somewhat biased by the performance and efficiency of the enzyme which can differ depending on the sample. WGBS uses physical shearing to chop the genome into 200-300 bp fragments which are amenable to NGS. The upside is that every fragment is theoretically sequenced, irrespective of CpG content, leading to potentially larger numbers of tumor specific biomarkers. The disadvantage is that most of the genome is not regulatory or suitable for biomarker discovery and so will be discarded. WGBS is also more costly to perform at a comparable depth of vertical coverage. The advent of higher capacity sequencers and flow cells, and the advance of NGS technology in general, has mitigated the cost to some degree—although it still remains less affordable than RRBS or other enrichment sequencing protocols. 
     Samples were identical to those used in the RRBS study to allow for direct comparison. Tissue and blood was obtained from Mayo Clinic biospecimen repositories with institutional IRB oversight. Samples were chosen with strict adherence to subject research authorization and inclusion/exclusion criteria. Cancers consisted of 21 metastatic melanomas. Controls included 15 non-neoplastic skin epidermis samples, 16 benign melanocytic nevi, and 36 whole blood derived leukocytes. Tissues were macro-dissected and histology reviewed by an expert pathologist. Samples were age matched, randomized, and blinded. DNA was purified using the QIAamp DNA Tissue Mini kit and QIAamp DNA Blood Mini kit (Qiagen, Valencia Calif.), respectively. DNA was re-purified with AMPure XP beads (Beckman-Coulter, Brea Calif.) and quantified by PicoGreen (Thermo-Fisher, Waltham Mass.). DNA integrity was assessed using qPCR. 
     Sequencing libraries were prepared following the method of Ulrich et al (Nature Protocols. 10, 475-483 (2015)) with modifications. Briefly 100 ng of DNA was sheared on a Covaris ultrasonicator to a 200 bp target size. Fragments &gt;600 bp were size selected away using 0.6× ampure beads (Beckman). The supernatant was then brought up to 1.4× to purify the remaining fragments with a lower limit of 100 bp. Ends were repaired with the End-It DNA end-repair kit (Epicentre) and purified with 1.4× Ampure beads, followed by a-tailing using Klenow (3′-5′ exo-(NEB) and a subsequent 1.4× purification. Methylated adapters (NEXTflex Bisulfite-Seq Barcodes—Bioo Scientific) were ligated with T4 DNA Ligase (NEB), bisulfite treated twice with the Epitect 96 Kit (Qiagen), and bead purified. Libraries were then tested by SYBR Green qPCR to determine optimal enrichment cycles—and then enriched by either 10,13,16, or 18 cycles using the KAPA HiFi HotStart Uracil+ReadyMix Kit (Kapa Biosystems). Enriched libraries were bead purified at a 1:1 ratio, indexed together in groups of 24, re-purified, and quantified on the Bioanalyzer 2100 (Agilent) and using qPCR (Kapa). Concentrations ranged from 5-80 nM. 
     Paired end 150 cycle sequencing was performed on the Nova-Seq using 1 S4 flow cell per 24 samples (Illumina). Reads were processed by Illumina pipeline modules for image analysis and base calling. Secondary analysis was performed using a WGBS modified version of SAAP-RRBS, a Mayo developed bioinformatics suite. Briefly, reads were cleaned-up using Trim-Galore and aligned to the GRCh37/hg19 reference genome build with BSMAP. Methylation ratios were determined by calculating C/(C+T) or conversely, G/(G+A) for reads mapping to reverse strand, for CpGs with coverage ≥5× and base quality score ≥20. 
     A proprietary DMR identification pipeline and regression package was used to derive DMRs based on average methylation values of the CpG. The difference in average methylation percentage was compared between malignant melanoma cases, tissue controls and buffy coat controls; a tiled reading frame within 100 base pairs of each mapped CpG was used to identify DMRs where control methylation was &lt;5%; DMRs were only analyzed if the total depth of coverage was 5 reads per subject on average and the variance across subgroups was &gt;0. 
     Following regression, DMRs were ranked by p-value, area under the receiver operating characteristic curve (AUC) and fold-change difference between cases and all controls. No adjustments for false discovery were made during this phase as independent validation was planned a priori. 
     Individual CpGs were ranked by hypermethylation ratio, namely the number of methylated cytosines at a given locus over the total cytosine count at that site. For cases, the ratios were required to be ≥0.20 (20%); for tissue controls, ≤0.05 (5%) tissue vs tissue analysis; ≥0.30 (30%) tissue vs buffy coat; for buffy coat controls, ≤0.01 (1%). CpGs which did not meet these criteria were discarded. Subsequently, candidate CpGs were binned by genomic location into DMRs (differentially methylated regions) ranging from approximately 30-300 bp with a minimum cut-off of 5 CpGs per region. DMRs with excessively high CpG density (&gt;30%) were excluded to avoid GC-related amplification problems in the validation phase. For each candidate region, a 2-D matrix was created which compared individual CpGs in a sample-to-sample fashion for both cases and controls. These CpG matrices were then compared back to the reference sequence to assess whether genomically contiguous methylation sites had been discarded during the initial filtering. From this subset of regions, final selections required coordinated and contiguous hypermethylation (in cases) of individual CpGs across the DMR sequence on a per sample level. Conversely, control samples had to have at least 10-fold less methylation than cases and the CpG pattern had to be more random and less coordinated. 
     Results 
     A modified WGBS methodology of sample preparation, sequencing, in combination with proprietary analyses pipelines and filters (outlined in Methods) was used to identify and narrow differentially methylated regions (DMRs) to those which would pinpoint these skin cancers and excel in a clinical testing environment. The WGBS DMRs showed substantial (roughly 50%) overlap with RRBS DMRs and the performance between the two studies correlated very well. However, there were many high performing biomarkers which were not seen with in the RRBS data. From the tissue-to-tissue analysis, 48 hypermethylated malignant melanomas (MM) DMRs were identified (Table 7A). All had AUCs&gt;0.90 and FC ratios &gt;10 (Table 7B). The tissue to leukocyte (buffy coat) analysis yielded 111 hypermethylated MM+epidermis tissue DMRs with less than 1% noise in WBCs (Table 8A). AUCs for all 111 were &gt;0.95 and FC ratios &gt;50 (Table 8B). The DMRs included in these tables were ones that did not appear in the RRBS study. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 7A 
               
               
                   
               
               
                   
                   
                   
                 DMR Start-End Positions 
               
               
                 DMR No. 
                 Gene Annotation 
                 Chromosome No. 
                 (GRCh37/hg19) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 163 
                 AGRN 
                 1 
                 969279-969321 
               
               
                 164 
                 BMP8B 
                 1 
                 40236184-40236211 
               
               
                 165 
                 C17orf46_B 
                 17 
                 43339390-43339782 
               
               
                 166 
                 C17orf57_B 
                 17 
                 45500815-45500840 
               
               
                 167 
                 C2CD4A 
                 15 
                 62359116-62359191 
               
               
                 168 
                 C2CD4D_B 
                 1 
                 151811304-151811332 
               
               
                 169 
                 CDYL 
                 6 
                 4775223-4775857 
               
               
                 170 
                 CMAH 
                 6.00E+00 
                 25137865-25137971 
               
               
                 171 
                 DENND2D 
                 1 
                 111747303-111747428 
               
               
                 172 
                 DLEU2 
                 13 
                 50701729-50701773 
               
               
                 173 
                 DSCR6 
                 21 
                 38378747-38378780 
               
               
                 174 
                 FLJ22536_B 
                 6 
                 21666257-21667030 
               
               
                 175 
                 FLJ45983 
                 10 
                 8097151-8097279 
               
               
                 176 
                 FOXF2 
                 6 
                 1393820-1393871 
               
               
                 177 
                 GALNT3_B 
                 2 
                 166650395-166650909 
               
               
                 178 
                 HCG4P6 
                 6 
                 29895017-29895100 
               
               
                 179 
                 HOXA9_B 
                 7 
                 27205761-27205801 
               
               
                 180 
                 KIFC2 
                 8 
                 145697900-145697946 
               
               
                 181 
                 LDLRAD2 
                 1 
                 22140884-22141031 
               
               
                 182 
                 LY75 
                 2 
                 160760468-160760605 
               
               
                 183 
                 LYL1 
                 19 
                 13210266-13210316 
               
               
                 184 
                 LYN 
                 8 
                 56791976-56792040 
               
               
                 185 
                 MAPK13 
                 6 
                 36098567-36098815 
               
               
                 186 
                 MAX.chr1.1072486-1072508 
                 1 
                 1072486-1072508 
               
               
                 187 
                 MAX.chr1.32237693-32237785 
                 1 
                 32237693-32237785 
               
               
                 188 
                 MAX.chr10.62492374-62492793 
                 10 
                 62492374-62492793 
               
               
                 189 
                 MAX.chr11.14926535-14926715 
                 11 
                 14926535-14926715 
               
               
                 190 
                 MAX.chr16.54970444-54970469 
                 16 
                 54970444-54970469 
               
               
                 191 
                 MAX.chr19.16439332-16439390 
                 19 
                 16439332-16439390 
               
               
                 192 
                 MAX.chr20.21080670-21081280 
                 20 
                 21080670-21081280 
               
               
                 193 
                 MAX.chr4.113432264-113432298 
                 4 
                 113432264-113432298 
               
               
                 194 
                 MAX.chr7.129425668-129425719 
                 7 
                 129425668-129425719 
               
               
                 195 
                 MAX.chr8.82543163-82543213 
                 8 
                 82543163-82543213 
               
               
                 196 
                 PARP15 
                 3 
                 122296541-122296587 
               
               
                 197 
                 PRKAG2 
                 7 
                 151329831-151329982 
               
               
                 198 
                 PROC_A 
                 2 
                 128173619-128173670 
               
               
                 199 
                 PROM1_B 
                 4 
                 16085259-16085484 
               
               
                 200 
                 PTGER4_A 
                 5 
                 40681512-40681876 
               
               
                 201 
                 PTP4A3 
                 8 
                 142428210-142428265 
               
               
                 202 
                 SDCCAG8 
                 1 
                 243646466-243646488 
               
               
                 203 
                 SH3PXD2A 
                 10 
                 105453184-105453230 
               
               
                 204 
                 SLC2A2 
                 3 
                 170746270-170746292 
               
               
                 205 
                 SLC35D3 
                 6 
                 137241799-137241883 
               
               
                 206 
                 TBR1 
                 2 
                 162271760-162271819 
               
               
                 207 
                 TBX2 
                 17 
                 59481777-59481811 
               
               
                 208 
                 TCP11 
                 6 
                 35108849-35108977 
               
               
                 209 
                 TFAP2A 
                 6 
                 10419530-10419646 
               
               
                 210 
                 TRIM73 
                 7 
                 75028743-75028905 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 7B 
               
               
                   
               
               
                   
                   
                 Area Under 
                   
                   
               
               
                 DMR No. 
                 Gene Annotation 
                 Curve 
                 Fold-Change 
                 p-value 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 163 
                 AGRN 
                 1 
                 33.01 
                 0.006078 
               
               
                 164 
                 BMP8B 
                 0.9643 
                 19.48 
                 0.005893 
               
               
                 165 
                 C17orf46_B 
                 0.9333 
                 25.6 
                 0.004365 
               
               
                 166 
                 C17orf57_B 
                 0.9464 
                 15.15 
                 0.003556 
               
               
                 167 
                 C2CD4A 
                 0.981 
                 35.75 
                 0.004804 
               
               
                 168 
                 C2CD4D_B 
                 1 
                 24.7 
                 0.004156 
               
               
                 169 
                 CDYL 
                 1 
                 48.54 
                 0.00356 
               
               
                 170 
                 CMAH 
                 1 
                 34.77 
                 2.81E−05 
               
               
                 171 
                 DENND2D 
                 0.9333 
                 17.63 
                 0.004753 
               
               
                 172 
                 DLEU2 
                 1 
                 44.81 
                 0.002166 
               
               
                 173 
                 DSCR6 
                 0.9567 
                 56.06 
                 0.008127 
               
               
                 174 
                 FLJ22536_B 
                 0.9619 
                 54.14 
                 0.008343 
               
               
                 175 
                 FLJ45983 
                 0.9333 
                 14.11 
                 0.009994 
               
               
                 176 
                 FOXF2 
                 0.9167 
                 17.63 
                 0.003168 
               
               
                 177 
                 GALNT3_B 
                 1 
                 112.6 
                 0.002757 
               
               
                 178 
                 HCG4P6 
                 0.9 
                 34.97 
                 0.008641 
               
               
                 179 
                 HOXA9_B 
                 1 
                 34.46 
                 0.001253 
               
               
                 180 
                 KIFC2 
                 0.9833 
                 92.67 
                 0.004278 
               
               
                 181 
                 LDLRAD2 
                 0.9286 
                 39.68 
                 0.003913 
               
               
                 182 
                 LY75 
                 0.9125 
                 11.68 
                 0.000547 
               
               
                 183 
                 LYL1 
                 0.9898 
                 14.97 
                 0.00264 
               
               
                 184 
                 LYN 
                 0.9619 
                 24.06 
                 0.006377 
               
               
                 185 
                 MAPK13 
                 1 
                 69.67 
                 0.001806 
               
               
                 186 
                 MAX.chr1.1072486-1072508 
                 0.9082 
                 51.23 
                 0.001488 
               
               
                 187 
                 MAX.chr1.32237693-32237785 
                 0.9833 
                 30.18 
                 0.004089 
               
               
                 188 
                 MAX.chr10.62492374-62492793 
                 0.9583 
                 11.59 
                 0.001119 
               
               
                 189 
                 MAX.chr11.14926535-14926715 
                 0.9833 
                 33.21 
                 0.001753 
               
               
                 190 
                 MAX.chr16.54970444-54970469 
                 0.9083 
                 25.38 
                 0.009217 
               
               
                 191 
                 MAX.chr19.16439332-16439390 
                 0.981 
                 25.69 
                 0.003778 
               
               
                 192 
                 MAX.chr20.21080670-21081280 
                 0.9125 
                 17.31 
                 0.003164 
               
               
                 193 
                 MAX.chr4.113432264-113432298 
                 0.9541 
                 35.56 
                 0.001857 
               
               
                 194 
                 MAX.chr7.129425668-129425719 
                 0.9429 
                 21.28 
                 0.006163 
               
               
                 195 
                 MAX.chr8.82543163-82543213 
                 0.9238 
                 23.35 
                 0.007549 
               
               
                 196 
                 PARP15 
                 1 
                 50.61 
                 0.002434 
               
               
                 197 
                 PRKAG2 
                 0.9667 
                 28.21 
                 0.001717 
               
               
                 198 
                 PROC_A 
                 0.9476 
                 28.81 
                 0.001883 
               
               
                 199 
                 PROM1_B 
                 0.9375 
                 40.17 
                 0.004806 
               
               
                 200 
                 PTGER4_A 
                 1 
                 33 
                 0.002816 
               
               
                 201 
                 PTP4A3 
                 0.9429 
                 20.28 
                 0.01 
               
               
                 202 
                 SDCCAG8 
                 0.9083 
                 27.26 
                 0.005528 
               
               
                 203 
                 SH3PXD2A 
                 0.9241 
                 53.51 
                 0.009754 
               
               
                 204 
                 SLC2A2 
                 0.95 
                 65.19 
                 0.005763 
               
               
                 205 
                 SLC35D3 
                 0.9292 
                 11.24 
                 0.003726 
               
               
                 206 
                 TBR1 
                 0.9042 
                 18.6 
                 0.003899 
               
               
                 207 
                 TBX2 
                 0.9333 
                 22.17 
                 0.007544 
               
               
                 208 
                 TCP11 
                 0.9898 
                 58.65 
                 0.001706 
               
               
                 209 
                 TFAP2A 
                 0.9087 
                 38.54 
                 0.007251 
               
               
                 210 
                 TRIM73 
                 1 
                 37.94 
                 0.005082 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
             
               
                 TABLE 8A 
               
               
                   
               
               
                   
                   
                   
                 DMR Start-End Positions 
               
               
                 DMR No. 
                 Gene Annotation 
                 Chromosome No. 
                 (GRCh37/hg19) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 211 
                 ABHD15 
                 17 
                 27893358-27893434 
               
               
                 212 
                 ACAP1 
                 17 
                 7240058-7240106 
               
               
                 213 
                 ACTB 
                 7 
                 5571593-5571712 
               
               
                 214 
                 ADRBK1 
                 11 
                 67035439-67036211 
               
               
                 215 
                 ANO9 
                 11 
                 441940-442058 
               
               
                 216 
                 ATP6V1B1 
                 2 
                 71192413-71192452 
               
               
                 217 
                 BEST4 
                 1 
                 45252148-45252191 
               
               
                 218 
                 C1orf177 
                 1 
                 55266723-55266832 
               
               
                 219 
                 CCDC140 
                 2 
                 223167358-223167385 
               
               
                 220 
                 CCND3 
                 6 
                 41908118-41908158 
               
               
                 221 
                 CD69 
                 12 
                 9917353-9917467 
               
               
                 222 
                 CDC42EP1 
                 22 
                 37962519-37962558 
               
               
                 223 
                 CHRM1 
                 11 
                 62693755-62693782 
               
               
                 224 
                 CSK 
                 15 
                 75069519-75069619 
               
               
                 225 
                 CYTH1 
                 17 
                 76771324-76771584 
               
               
                 226 
                 DDN 
                 12 
                 49391122-49391209 
               
               
                 227 
                 DEDD2 
                 19 
                 42703648-42703891 
               
               
                 228 
                 DGKZ 
                 11 
                 46367778-46367847 
               
               
                 229 
                 DHH 
                 12 
                 49483661-49483738 
               
               
                 230 
                 DLG5 
                 10 
                 79633792-79633845 
               
               
                 231 
                 DNM2 
                 19 
                 10870312-10870427 
               
               
                 232 
                 DNMT3A 
                 2 
                 25499764-25499804 
               
               
                 233 
                 DUSP2 
                 2 
                 96812221-96812268 
               
               
                 234 
                 ESRRG 
                 1 
                 217308088-217308118 
               
               
                 235 
                 FBRSL1 
                 12 
                 133065476-133065776 
               
               
                 236 
                 FOXP1 
                 3 
                 71630818-71630894 
               
               
                 237 
                 FOXP4_A 
                 6 
                 41515929-41515972 
               
               
                 238 
                 FOXP4_B 
                 6 
                 41528310-41528503 
               
               
                 239 
                 FRZB 
                 2 
                 183731563-183731600 
               
               
                 240 
                 GALNT3_C 
                 2 
                 166650352-166650402 
               
               
                 241 
                 GATA2 
                 3 
                 128211626-128211681 
               
               
                 242 
                 GJB2 
                 13 
                 20767909-20768079 
               
               
                 243 
                 GNG7 
                 19 
                 2620764-2620859 
               
               
                 244 
                 GP5_A 
                 3 
                 194117873-194117897 
               
               
                 245 
                 GP5_B 
                 3 
                 194118511-194118559 
               
               
                 246 
                 GP5_C 
                 3 
                 194118799-194118838 
               
               
                 247 
                 GPR132 
                 14 
                 105527858-105527909 
               
               
                 248 
                 GPR135_B 
                 14 
                 59931142-59931177 
               
               
                 249 
                 HAAO 
                 2 
                 43019992-43020017 
               
               
                 250 
                 HOXB5_C 
                 17 
                 46671011-46671084 
               
               
                 251 
                 HOXB5_D 
                 17 
                 46673976-46674007 
               
               
                 252 
                 HSF5 
                 17 
                 56565263-56565284 
               
               
                 253 
                 ICAM2 
                 17 
                 62097597-62097723 
               
               
                 254 
                 IFFO2 
                 1 
                 19250393-19250485 
               
               
                 255 
                 KCNH3 
                 12 
                 49943015-49943046 
               
               
                 256 
                 KCNQ4_B 
                 1 
                 41284305-41284411 
               
               
                 257 
                 KIAA0182 
                 16 
                 85649545-85649580 
               
               
                 258 
                 KLHDC7B 
                 22 
                 50987206-50987271 
               
               
                 259 
                 KREMEN1_B 
                 22 
                 29467754-29467795 
               
               
                 260 
                 LHFPL2 
                 5 
                 77805840-77805876 
               
               
                 261 
                 LIMD1 
                 3 
                 45706043-45706236 
               
               
                 262 
                 LOC440925 
                 2 
                 171570298-171570337 
               
               
                 263 
                 MAP3K5 
                 6 
                 137112514-137112610 
               
               
                 264 
                 MAX.chr1.203256204-203256316 
                 1 
                 203256204-203256316 
               
               
                 265 
                 MAX.chr1.203258714-203258909 
                 1 
                 203258714-203258909 
               
               
                 266 
                 MAX.chr1.22366401-22366546 
                 1 
                 22366401-22366546 
               
               
                 267 
                 MAX.chr1.54941184-54941237 
                 1 
                 54941184-54941237 
               
               
                 268 
                 MAX.chr11.14927004-14927275 
                 11 
                 14927004-14927275 
               
               
                 269 
                 MAX.chr12.30976208-30976253 
                 12 
                 30976208-30976253 
               
               
                 270 
                 MAX.chr15.31556237-31556534 
                 15 
                 31556237-31556534 
               
               
                 271 
                 MAX.chr19.41834559-41834634 
                 19 
                 41834559-41834634 
               
               
                 272 
                 MAX.chr19.50003691-50003720 
                 19 
                 50003691-50003720 
               
               
                 273 
                 MAX.chr2.69135655-69135781 
                 2 
                 69135655-69135781 
               
               
                 274 
                 MAX.chr21.37670549-37670625 
                 21 
                 37670549-37670625 
               
               
                 275 
                 MAX.chr22.36848164-36848205 
                 22 
                 36848164-36848205 
               
               
                 276 
                 MAX.chr4.186050036-186050085 
                 4 
                 186050036-186050085 
               
               
                 277 
                 MAX.chr5.42992599-42992772 
                 5 
                 42992599-42992772 
               
               
                 278 
                 MAX.chr8.101822019-101822077 
                 8 
                 101822019-101822077 
               
               
                 279 
                 MAX.chr8.80695857-80695919 
                 8 
                 80695857-80695919 
               
               
                 280 
                 MAX.chr8.80803443-80803495 
                 8 
                 80803443-80803495 
               
               
                 281 
                 MAX.chr9.139595896-139596026 
                 9 
                 139595896-139596026 
               
               
                 282 
                 MORN3 
                 12 
                 122096796-122096850 
               
               
                 283 
                 MPZ_B 
                 1 
                 161275414-161275553 
               
               
                 284 
                 NDRG4 
                 16 
                 58535208-58535288 
               
               
                 285 
                 NFATC2_B 
                 20 
                 50158872-50159008 
               
               
                 286 
                 OTX1 
                 2 
                 63283968-63284002 
               
               
                 287 
                 PAMR1 
                 11 
                 35547114-35547153 
               
               
                 288 
                 PDLIM2_A 
                 8 
                 22437870-22438005 
               
               
                 289 
                 PDLIM2_B 
                 8 
                 22438128-22438191 
               
               
                 290 
                 PHF20 
                 20 
                 34356073-34356200 
               
               
                 291 
                 PPFIA4 
                 1 
                 203044814-203044863 
               
               
                 292 
                 PROC_B 
                 2 
                 128173738-128173906 
               
               
                 293 
                 PRR15 
                 7 
                 29603319-29603434 
               
               
                 294 
                 PSTPIP1 
                 15 
                 77287762-77287907 
               
               
                 295 
                 PTGER4_B 
                 5 
                 40681167-40681229 
               
               
                 296 
                 PTK2B 
                 8 
                 27183885-27184171 
               
               
                 297 
                 PTPN6 
                 12 
                 7060368-7060475 
               
               
                 298 
                 RGS3 
                 9 
                 116342944-116343115 
               
               
                 299 
                 RIN2 
                 20 
                 19917988-19918066 
               
               
                 300 
                 RNF207_B 
                 1 
                 6265952-6265974 
               
               
                 301 
                 RNF207_C 
                 1 
                 6266219-6266258 
               
               
                 302 
                 RNF220_B 
                 1 
                 44874701-44874823 
               
               
                 303 
                 RNF44 
                 5 
                 175960664-175960896 
               
               
                 304 
                 RPS6KA1 
                 1 
                 26868823-26868917 
               
               
                 305 
                 RPTOR 
                 17 
                 78638920-78639074 
               
               
                 306 
                 SELPLG 
                 12 
                 109029388-109029607 
               
               
                 307 
                 SGK1 
                 6 
                 134561914-134562045 
               
               
                 308 
                 SLC2A1 
                 1 
                 43397754-43398090 
               
               
                 309 
                 SMARCA4 
                 19 
                 11159902-11160083 
               
               
                 310 
                 SPTBN1 
                 2 
                 54785828-54785886 
               
               
                 311 
                 SYN3 
                 22 
                 33023163-33023242 
               
               
                 312 
                 SYTL1 
                 1 
                 27669462-27669641 
               
               
                 313 
                 TFAP2E 
                 1 
                 36042961-36043015 
               
               
                 314 
                 TFEB 
                 6 
                 41675581-41675635 
               
               
                 315 
                 THAP4 
                 2 
                 242549862-242549967 
               
               
                 316 
                 TOX 
                 8 
                 60030355-60030405 
               
               
                 317 
                 TRABD 
                 22 
                 50629861-50630029 
               
               
                 318 
                 TRH 
                 3 
                 129693636-129693699 
               
               
                 319 
                 TXNRD2 
                 22 
                 19879091-19879295 
               
               
                 320 
                 UST 
                 6 
                 149082648-149082945 
               
               
                 321 
                 WDR66 
                 12 
                 122356325-122356391 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 8B 
               
               
                   
               
               
                   
                   
                 Area Under 
                   
                   
               
               
                 DMR No. 
                 Gene Annotation 
                 Curve 
                 Fold-Change 
                 p-value 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 211 
                 ABHD15 
                 0.9667 
                 7.88E+08 
                 0.995 
               
               
                 212 
                 ACAP1 
                 1 
                 1.82E+09 
                 0.9929 
               
               
                 213 
                 ACTB 
                 0.9667 
                 1.51E+09 
                 0.9954 
               
               
                 214 
                 ADRBK1 
                 1 
                 198.6 
                 0.0004594 
               
               
                 215 
                 ANO9 
                 1 
                 1.27E+09 
                 0.9951 
               
               
                 216 
                 ATP6V1B1 
                 0.9667 
                 8.37E+08 
                 0.9949 
               
               
                 217 
                 BEST4 
                 0.95 
                  78.09 
                 0.007705 
               
               
                 218 
                 C1orf177 
                 0.9643 
                 7E+08 
                 0.9949 
               
               
                 219 
                 CCDC140 
                 1 
                 199.8 
                 0.009637 
               
               
                 220 
                 CCND3 
                 1 
                 8.02E+08 
                 0.9956 
               
               
                 221 
                 CD69 
                 0.9533 
                 152.9 
                 0.00634 
               
               
                 222 
                 CDC42EP1 
                 1 
                 251.6 
                 9.06E−05 
               
               
                 223 
                 CHRM1 
                 0.9643 
                 6.03E+08 
                 0.9949 
               
               
                 224 
                 CSK 
                 0.9667 
                  4.6E+08 
                 0.9932 
               
               
                 225 
                 CYTH1 
                 1 
                 2.66E+09 
                 0.9936 
               
               
                 226 
                 DDN 
                 0.9667 
                 1.15E+09 
                 0.9952 
               
               
                 227 
                 DEDD2 
                 1 
                 608.8 
                 0.0006947 
               
               
                 228 
                 DGKZ 
                 1 
                 1.90E+09 
                 0.994 
               
               
                 229 
                 DHH 
                 1 
                 5.72E+08 
                 0.9937 
               
               
                 230 
                 DLG5 
                 0.9733 
                  73.42 
                 0.002512 
               
               
                 231 
                 DNM2 
                 1 
                 104.5 
                 0.001014 
               
               
                 232 
                 DNMT3A 
                 1 
                 4.06E+09 
                 0.9948 
               
               
                 233 
                 DUSP2 
                 1 
                 172.6 
                 0.006556 
               
               
                 234 
                 ESRRG 
                 0.9667 
                 2.04E+08 
                 0.9944 
               
               
                 235 
                 FBRSL1 
                 1 
                 136.1 
                 0.007791 
               
               
                 236 
                 FOXP1 
                 0.9667 
                 2.16E+08 
                 0.9933 
               
               
                 237 
                 FOXP4_A 
                 0.9519 
                 201.6 
                 0.00375 
               
               
                 238 
                 FOXP4_B 
                 1 
                 3.88E+08 
                 0.9941 
               
               
                 239 
                 FRZB 
                 0.9667 
                 1.38E+09 
                 0.9949 
               
               
                 240 
                 GALNT3_C 
                 0.9643 
                 1.19E+09 
                 0.9948 
               
               
                 241 
                 GATA2 
                 0.9667 
                 7.78E+08 
                 0.9948 
               
               
                 242 
                 GJB2 
                 1 
                 1.51E+09 
                 0.9952 
               
               
                 243 
                 GNG7 
                 0.9667 
                 1.87E+09 
                 0.9942 
               
               
                 244 
                 GP5_A 
                 1 
                 2.13E+09 
                 0.9934 
               
               
                 245 
                 GP5_B 
                 0.9567 
                 197.3 
                 0.005652 
               
               
                 246 
                 GP5_C 
                 1 
                 130.6 
                 0.00432 
               
               
                 247 
                 GPR132 
                 1 
                 1.17E+09 
                 0.9941 
               
               
                 248 
                 GPR135_B 
                 0.9643 
                 6.89E+08 
                 0.9958 
               
               
                 249 
                 HAAO 
                 0.9615 
                  2.6E+08 
                 0.9935 
               
               
                 250 
                 HOXB5_C 
                 0.96 
                 207.2 
                 0.009996 
               
               
                 251 
                 HOXB5_D 
                 0.9933 
                 222.6 
                 0.004066 
               
               
                 252 
                 HSF5 
                 1 
                 615.6 
                 0.0015 
               
               
                 253 
                 ICAM2 
                 0.9667 
                 6.27E+08 
                 0.9942 
               
               
                 254 
                 IFFO2 
                 0.9567 
                  82.21 
                 0.008566 
               
               
                 255 
                 KCNH3 
                 1 
                 116.9 
                 0.000725 
               
               
                 256 
                 KCNQ4_B 
                 0.9643 
                 8.78E+08 
                 0.9953 
               
               
                 257 
                 KIAA0182 
                 0.9556 
                 112.2 
                 0.008084 
               
               
                 258 
                 KLHDC7B 
                 0.9667 
                 5.54E+08 
                 0.9941 
               
               
                 259 
                 KREMEN1_B 
                 0.9667 
                 8.69E+08 
                 0.9939 
               
               
                 260 
                 LHFPL2 
                 0.95 
                 105.9 
                 0.005347 
               
               
                 261 
                 LIMD1 
                 1 
                 278.2 
                 0.008128 
               
               
                 262 
                 LOC440925 
                 1 
                 1.31E+09 
                 0.9948 
               
               
                 263 
                 MAP3K5 
                 0.9667 
                 4.22E+08 
                 0.9931 
               
               
                 264 
                 MAX.chr1.203256204-203256316 
                 0.9933 
                 107.5 
                 0.001409 
               
               
                 265 
                 MAX.chr1.203258714-203258909 
                 1 
                 304.1 
                 0.0091 
               
               
                 266 
                 MAX.chr1.22366401-22366546 
                 1 
                 1.40E+09 
                 0.9954 
               
               
                 267 
                 MAX.chr1.54941184-54941237 
                 1 
                 4.76E+08 
                 0.9928 
               
               
                 268 
                 MAX.chr11.14927004-14927275 
                 1 
                 434.4 
                 0.007614 
               
               
                 269 
                 MAX.chr12.30976208-30976253 
                 1 
                 282.8 
                 0.007978 
               
               
                 270 
                 MAX.chr15.31556237-31556534 
                 1 
                 152.8 
                 0.006391 
               
               
                 271 
                 MAX.chr19.41834559-41834634 
                 1 
                 233.6 
                 0.009335 
               
               
                 272 
                 MAX.chr19.50003691-50003720 
                 0.9667 
                 1.64E+09 
                 0.9955 
               
               
                 273 
                 MAX.chr2.69135655-69135781 
                 1 
                  99.2 
                 0.008221 
               
               
                 274 
                 MAX.chr21.37670549-37670625 
                 1 
                 1.21E+09 
                 0.9955 
               
               
                 275 
                 MAX.chr22.36848164-36848205 
                 0.9867 
                 137.2 
                 0.005796 
               
               
                 276 
                 MAX.chr4.186050036-186050085 
                 1 
                  68.76 
                 0.0057 
               
               
                 277 
                 MAX.chr5.42992599-42992772 
                 0.9667 
                  2.6E+08 
                 0.9937 
               
               
                 278 
                 MAX.chr8.101822019-101822077 
                 0.9567 
                 312.5 
                 0.008165 
               
               
                 279 
                 MAX.chr8.80695857-80695919 
                 1 
                 4.45E+08 
                 0.9935 
               
               
                 280 
                 MAX.chr8.80803443-80803495 
                 1 
                 2.32E+08 
                 0.9938 
               
               
                 281 
                 MAX.chr9.139595896-139596026 
                 0.96 
                 133.8 
                 0.001154 
               
               
                 282 
                 MORN3 
                 1 
                 9.83E+08 
                 0.9946 
               
               
                 283 
                 MPZ_B 
                 1 
                 8.55E+08 
                 0.9943 
               
               
                 284 
                 NDRG4 
                 0.9667 
                 4.43E+08 
                 0.9929 
               
               
                 285 
                 NFATC2_B 
                 1 
                 2.06E+09 
                 0.9937 
               
               
                 286 
                 OTX1 
                 0.9923 
                 258.6 
                 0.001903 
               
               
                 287 
                 PAMR1 
                 0.9667 
                 6.26E+08 
                 0.9944 
               
               
                 288 
                 PDLIM2_A 
                 1 
                 1.59E+09 
                 0.9951 
               
               
                 289 
                 PDLIM2_B 
                 1 
                 389.6 
                 0.008777 
               
               
                 290 
                 PHF20 
                 0.9643 
                 1.87E+08 
                 0.9943 
               
               
                 291 
                 PPFIA4 
                 1 
                 1.66E+09 
                 0.9945 
               
               
                 292 
                 PROC_B 
                 0.95 
                 188.7 
                 0.002602 
               
               
                 293 
                 PRR15 
                 0.9567 
                 122.9 
                 0.002215 
               
               
                 294 
                 PSTPIP1 
                 1 
                 308.6 
                 0.008096 
               
               
                 295 
                 PTGER4_B 
                 0.9643 
                 9.54E+08 
                 0.9953 
               
               
                 296 
                 PTK2B 
                 1 
                 242.6 
                 0.003993 
               
               
                 297 
                 PTPN6 
                 1 
                 4.84E+09 
                 0.995 
               
               
                 298 
                 RGS3 
                 0.9567 
                  87.52 
                 0.001445 
               
               
                 299 
                 RIN2 
                 1 
                 607.6 
                 0.00234 
               
               
                 300 
                 RNF207_B 
                 0.9643 
                 1.07E+09 
                 0.9941 
               
               
                 301 
                 RNF207_C 
                 1 
                 1.68E+09 
                 0.9946 
               
               
                 302 
                 RNF220_B 
                 0.9733 
                  56.55 
                 0.008502 
               
               
                 303 
                 RNF44 
                 0.9667 
                 8.01E+08 
                 0.9936 
               
               
                 304 
                 RPS6KA1 
                 1 
                 206.1 
                 0.0009877 
               
               
                 305 
                 RPTOR 
                 1 
                 1.05E+09 
                 0.9946 
               
               
                 306 
                 SELPLG 
                 1 
                 211.9 
                 0.001638 
               
               
                 307 
                 SGK1 
                 1 
                  76.59 
                 0.005985 
               
               
                 308 
                 SLC2A1 
                 1 
                 121.9 
                 0.0002754 
               
               
                 309 
                 SMARCA4 
                 1 
                 474.8 
                 0.001605 
               
               
                 310 
                 SPTBN1 
                 0.9667 
                 4.93E+08 
                 0.9946 
               
               
                 311 
                 SYN3 
                 0.9667 
                 1.36E+09 
                 0.9948 
               
               
                 312 
                 SYTL1 
                 0.9533 
                 223.1 
                 0.00105 
               
               
                 313 
                 TFAP2E 
                 0.9933 
                 208.2 
                 0.004403 
               
               
                 314 
                 TFEB 
                 1 
                 9.96E+08 
                 0.9938 
               
               
                 315 
                 THAP4 
                 0.9667 
                 9.94E+08 
                 0.9953 
               
               
                 316 
                 TOX 
                 0.9667 
                 1.18E+09 
                 0.9949 
               
               
                 317 
                 TRABD 
                 1 
                 1.93E+09 
                 0.993 
               
               
                 318 
                 TRH 
                 1 
                 7.79E+08 
                 0.9939 
               
               
                 319 
                 TXNRD2 
                 1 
                 2.90E+09 
                 0.9951 
               
               
                 320 
                 UST 
                 1 
                 147.7 
                 0.002184 
               
               
                 321 
                 WDR66 
                 0.9917 
                 175.4 
                 0.004255 
               
               
                   
               
            
           
         
       
     
     TELQAS Oligonucleotides 
     Target enrichment long-probe quantitative amplified signal (TELQAS) (see, Kisiel J B, et al., Hepatology. 2018 Aug. 31) oligos (forward invasive primer, reverse primer, flap probe) were designed to CpG motifs within several of the markers as shown in  FIG.  1    and Table 9 for use in, for example, detecting the presence or absence of such markers in tissue and plasma samples. Table 10 refers to primers and Table 11 refers to probes configured for use in a TELQAS assay. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 9 
               
               
                   
               
               
                   
                   
                   
                 DMR Start-End Positions 
               
               
                 DMR No. 
                 Gene Annotation 
                 Chromosome No. 
                 (GRCh37/hg19) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 322 
                 MAX.chr10.62492680-62492822 
                 10 
                 62492680-62492822 
               
               
                 323 
                 TMEM30B_B 
                 14 
                 61747319-61747519 
               
               
                 324 
                 MAX.chr11:14926602-14926831 
                 11 
                 14926602-14926831 
               
               
                 325 
                 HOXA9_9650 
                 7 
                 27209650-27209717 
               
               
                 326 
                 C10orf55 B 
                 10 
                 75670643-75670799 
               
               
                 73 
                 MAX.chr17.73073682-73073830 
                 17 
                 73073682-73073830 
               
               
                 122 
                 TSPAN33 
                 7 
                 128809044-128809129 
               
               
                 327 
                 FAM174B_C 
                 15 
                 93199025-93199119 
               
               
                 86 
                 MAX.chr5.60921627-60921853 
                 5 
                 60921627-60921853 
               
               
                 111 
                 SIX4_A 
                 14 
                 61188480-61188614 
               
               
                 49 
                 KCNQ4_A 
                 1 
                 41284410-41284590 
               
               
                 32 
                 FOXP1 
                 3 
                 71630795-71630964 
               
               
                 105 
                 OXT 
                 20 
                 3052495-3052618 
               
               
                 328 
                 C2orf82_B 
                 2 
                 233740969-233741417 
               
               
                 329 
                 TAL1_B 
                 1 
                 47698041-47698147 
               
               
                 330 
                 BTBD19_B 
                 1 
                 45279358-45279574 
               
               
                 331 
                 MAX.chr10.22541874-22541948 
                 10 
                 22541874-22541948 
               
               
                 99 
                 MIR155HG 
                 21 
                 26934273-26934633 
               
               
                 85 
                 MAX.chr5.42992655-42992768 
                 5 
                 42992655-42992768 
               
               
                 97 
                 ME3 
                 11 
                 86382754-86383237 
               
               
                 45 
                 HOPX 
                 4 
                 57522436-57522653 
               
               
                 81 
                 MAX.chr20.3229151-3229791 
                 20 
                 3229151-3229791 
               
               
                 21 
                 CLIC5 
                 6 
                 45982945-45983289 
               
               
                 66 
                 MAX.chr13.29106812-29106917 
                 13 
                 29106812-29106917 
               
               
                 16 
                 FOXL2NB 
                 3 
                 138663981-138664076 
               
               
                 136 
                 FLJ22536_A 
                 6 
                 21666448-21666575 
               
               
                 146 
                 MAX.chr1.110627096-110627364 
                 1 
                 110627096-110627364 
               
               
                 151 
                 MAX.chr2.71116033-71116122 
                 2 
                 71116033-71116122 
               
               
                 152 
                 MAX.chr20.21080958-21081038 
                 20 
                 21080958-21081038 
               
               
                 153 
                 MAX.chr7.155259597-155259763 
                 7 
                 1552596597-155259763  
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 10 
               
               
                   
               
               
                   
                   
                 Seq 
                   
                 Seq 
                   
               
               
                 DMR 
                   
                 ID 
                 Forward Primer 5′-3′ 
                 ID 
                 Reverse Primer 5′-3′ 
               
               
                 No. 
                 Gene Annotation 
                 No. 
                 Sequence 
                 No. 
                 Sequence 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 322 
                 MAX.chr10.62492 
                 81 
                 TCGGGATAGAGGCGAG 
                 82 
                 GAAACCCGCTTTTCTT 
               
               
                   
                 680-62492822 
                   
                 AGTTC 
                   
                 TTTCCAAAC 
               
               
                   
               
               
                 323 
                 TMEM30B_B 
                 83 
                 CGAGTGCGTTTTTTATTA 
                 84 
                 GTTAAAAAAACTATTA 
               
               
                   
                   
                   
                 GCGTAGC 
                   
                 ACGATAACGCCGC 
               
               
                   
               
               
                 324 
                 MAX.chr11:14926 
                 85 
                 GGTTCGAAGGTATAGTG 
                 86 
                 AAAACCCACCGAATC 
               
               
                   
                 602-14926831 
                   
                 AGTTTCGTC 
                   
                 CTTCGA 
               
               
                   
               
               
                 325 
                 HOXA9_9650 
                 87 
                 TTCGTTCGTCGGGGCG 
                 88 
                 GTTTCCTACTTACCAA 
               
               
                   
                   
                   
                   
                   
                 AATAAAAAAAACGAAA 
               
               
                   
               
               
                 326 
                 C10orf55_B 
                 89 
                 TGTTGGGTTTTTTTCGTT 
                 90 
                 GAACCCCGCGTACTT 
               
               
                   
                   
                   
                 GGAGATC 
                   
                 CCG 
               
               
                   
               
               
                 73 
                 MAX.chr17.73073 
                 35 
                 TTTTTCGAGTCGTTTTAT 
                 36 
                 GAACTCCGAACGCCG 
               
               
                   
                 682-73073830 
                   
                 TTCGCGG 
                   
                 CTTAAACGTA 
               
               
                   
               
               
                 73 
                 MAX.chr17.73073 
                 91 
                 CGCGGTTATGGTTAGTA 
                 92 
                 GAACGACGCGAACTC 
               
               
                   
                 682-73073830 
                   
                 GCGGC 
                   
                 CGA 
               
               
                   
               
               
                 122 
                 TSPAN33 
                 79 
                 CGGTTGTAGGGAGGATT 
                 80 
                 ACGCCAAAAAACCCA 
               
               
                   
                   
                   
                 TCGAGGAAGTTC 
                   
                 ACGCA 
               
               
                   
               
               
                 122 
                 TSPAN33 
                 93 
                 GGCGTTAGGAGGTTTAG 
                 94 
                 CTCCAAAACCTTCTC 
               
               
                   
                   
                   
                 CGTATC 
                   
                 CCTAAATCGA 
               
               
                   
               
               
                 327 
                 FAM174B_C 
                 95 
                 AGTTCGCGTTATCGTCG 
                 96 
                 CCGCCCACGTAAAAC 
               
               
                   
                   
                   
                 TCG 
                   
                 CG 
               
               
                   
               
               
                 86 
                 MAX.chr5.609216 
                 43 
                 TCGTAGAGGTTATCGGA 
                 44 
                 AATACCCGAACCACA 
               
               
                   
                 27-60921853 
                   
                 TATAGCGA 
                   
                 AAACCCGCC 
               
               
                   
               
               
                 86 
                 MAX.chr5.609216 
                 97 
                 GTTTTAGAGGTCGTTAA 
                 98 
                 ATAAAACCGCAAAAA 
               
               
                   
                 27-60921853 
                   
                 GTTTCGGC 
                   
                 CCACCGA 
               
               
                   
               
               
                 111 
                 SIX4_A 
                 67 
                 GGTGGTTCGGCGTAATT 
                 68 
                 CGTACGCCTTCCGCA 
               
               
                   
                   
                   
                 AAAGCGT 
                   
                 AATATACGAA 
               
               
                   
               
               
                 111 
                 SIX4_A 
                 99 
                 TTTCGTTGGTTTTCGAG 
                 100 
                 CGAAATATACGTACG 
               
               
                   
                   
                   
                 CGC 
                   
                 CCTTCCGC 
               
               
                   
               
               
                 49 
                 KCNQ4_A 
                 23 
                 GGGCGAGGTTAGAGGG 
                 24 
                 GCCTCCTACTAAAAC 
               
               
                   
                   
                   
                 GTTGTTHC 
                   
                 TCCAACCCCCG 
               
               
                   
               
               
                 49 
                 KCNQ4_A 
                 101 
                 GTAGGAGGCGAGGTTTA 
                 102 
                 CCCGCCCCCTAATCC 
               
               
                   
                   
                   
                 AGCG 
                   
                 G 
               
               
                   
               
               
                 32 
                 FOXP1 
                 13 
                 GGGGCGATTTTTAGTAA 
                 14 
                 CTACCCTCTCTATCCT 
               
               
                   
                   
                   
                 GTTTTTTTCGT 
                   
                 AACCCCTCGAC 
               
               
                   
               
               
                 32 
                 FOXP1 
                 103 
                 GGTTCGTTCGTTCGTTC 
                 104 
                 AAAACTTACTAAAAAT 
               
               
                   
                   
                   
                 GTC 
                   
                 CGCCCCGA 
               
               
                   
               
               
                 105 
                 OXT 
                 61 
                 CGTTTGAGAATTTTAGG 
                 62 
                 CGAAACAACGAAACT 
               
               
                   
                   
                   
                 AGTTGAGCGG 
                   
                 AAAACGCGCT 
               
               
                   
               
               
                 105 
                 OXT 
                 105 
                 ATTTTGACGTTTCGTTTT 
                 106 
                 GAAACAACGAAACTA 
               
               
                   
                   
                   
                 TGATCGC 
                   
                 AAACGCGC 
               
               
                   
               
               
                 328 
                 C2orf82_B 
                 107 
                 TCGGCGTTATCGCGGTT 
                 108 
                 AAAACAAAAAACTTTC 
               
               
                   
                   
                   
                 ATC 
                   
                 TCAACGCGA 
               
               
                   
               
               
                 329 
                 TAL1_B 
                 109 
                 AAGGTATTGTCGCGGGT 
                 110 
                 AATCCCCACTCCCTC 
               
               
                   
                   
                   
                 TCG 
                   
                 CGA 
               
               
                   
               
               
                 330 
                 BTBD19_B 
                 111 
                 TGTTTTTTAAATGATTTA 
                 112 
                 CGAACGCCGAACACT 
               
               
                   
                   
                   
                 ACGTCGGGATTC 
                   
                 TCGA 
               
               
                   
               
               
                 331 
                 MAX.chr10.22541 
                 113 
                 CGGAATTTCGGTTTTCG 
                 114 
                 CCGAAAAACTTTCAAA 
               
               
                   
                 874-22541948 
                   
                 CGG 
                   
                 CACTAACCG 
               
               
                   
               
               
                 99 
                 MIR155HG 
                 53 
                 CGGATAGCGGAGTTTCG 
                 54 
                 AAACGTCTCCTTAATT 
               
               
                   
                   
                   
                 AGTCGTTC 
                   
                 CCCCGCGCTT 
               
               
                   
               
               
                 99 
                 MIR155HG 
                 55 
                 TAAGCGCGGGGAATTAA 
                 56 
                 CGAAAACGCGAAACT 
               
               
                   
                   
                   
                 GGAGACGT 
                   
                 AAAATCGACGTA 
               
               
                   
               
               
                 99 
                 MIR155HG 
                 115 
                 GGAGCGGATAGCGGAG 
                 116 
                 ACGAAAACGCGAAAC 
               
               
                   
                   
                   
                 TTTC 
                   
                 TAAAATCGA 
               
               
                   
               
               
                 85 
                 MAX.chr5.429926 
                 41 
                 GTTGGGGAAGTTTCGAA 
                 42 
                 TCACCCCAACTAAAA 
               
               
                   
                 55-42992768 
                   
                 TTTTTTAGATC 
                   
                 AAACTCCGAA 
               
               
                   
               
               
                 85 
                 MAX.chr5.429926 
                 116 
                 GGGGCGATAGTATTTAG 
                 117 
                 GATCTCAACACAACT 
               
               
                   
                 55-42992768 
                   
                 GTCGTCGA 
                   
                 CGTTACTCGA 
               
               
                   
               
               
                 97 
                 ME3 
                 51 
                 GTTGCGGGGTATCGGGT 
                 52 
                 CCTACCCTCTCCCAA 
               
               
                   
                   
                   
                 TTGATTC 
                   
                 AAAACGCGTC 
               
               
                   
               
               
                 97 
                 ME3 
                 118 
                 GGTTTTTGGTGATATCG 
                 119 
                 GACTAACTCCCTAAC 
               
               
                   
                   
                   
                 TATTCGCG 
                   
                 CGAACCG 
               
               
                   
               
               
                 45 
                 HOPX 
                 17 
                 TTGTATTTTTGTTTGCGA 
                 18 
                 CCGAAAATCGATAAA 
               
               
                   
                   
                   
                 CGGGGGC 
                   
                 AACCCGCGAA 
               
               
                   
               
               
                 45 
                 HOPX 
                 119 
                 CGGGGGCGAGATAGAT 
                 120 
                 TCTCAAAATCACCCC 
               
               
                   
                   
                   
                 GATTTC 
                   
                 CGCG 
               
               
                   
               
               
                 81 
                 MAX.chr20.32291 
                 121 
                 GCGTGGTTTTTATATAG 
                 122 
                 CGCCGTACACGAATA 
               
               
                   
                 51-3229791 
                   
                 TTCGGTCG 
                   
                 CCGA 
               
               
                   
               
               
                 21 
                 CLIC5 
                 7 
                 GGCGTTGGTTTGCGGAA 
                 8 
                 CACCCACCAAAACTC 
               
               
                   
                   
                   
                 AGGTTAC 
                   
                 GAAATACGCT 
               
               
                   
               
               
                 21 
                 CLIC5 
                 123 
                 GGAGTTTCGTAGCGGGC 
                 124 
                 CCTCCCAAAAAAACG 
               
               
                   
                   
                   
                 G 
                   
                 ACGCG 
               
               
                   
               
               
                 66 
                 MAX.chr13.29106 
                 33 
                 CGGTTTTGATTTTAGGG 
                 34 
                 TTAACTATCGAAAACG 
               
               
                   
                 812-29106917 
                   
                 CGT 
                   
                 ATTTCCGCGAA 
               
               
                   
               
               
                 66 
                 MAX.chr13.29106 
                 125 
                 GGAATGGTTTCGTAGTT 
                 126 
                 CGAATTAACTATCGAA 
               
               
                   
                 812-29106917 
                   
                 GCGC 
                   
                 AACGATTTCCGC 
               
               
                   
               
               
                 16 
                 FOXL2NB 
                 5 
                 TATGGATTGTACGGTAG 
                 6 
                 ACGCAAACCTCTTAA 
               
               
                   
                   
                   
                 TCGGGCGG 
                   
                 CCTTCTCTCACAAATC 
               
               
                   
                   
                   
                   
                   
                 G 
               
               
                   
               
               
                 16 
                 FOXL2NB 
                 126 
                 GGTCGGTGCGTTCGTTT 
                 127 
                 CACGCGTCTAACCAT 
               
               
                   
                   
                   
                 TTC 
                   
                 AAACTACAC 
               
               
                   
               
               
                 136 
                 FLJ22536_A 
                 11 
                 GAGTTTGGTAGTGGGAG 
                 12 
                 CCGAAACGAACTAAA 
               
               
                   
                   
                   
                 GAGATCGT 
                   
                 ACTACTCGAT 
               
               
                   
               
               
                 136 
                 FLJ22536_A 
                 127 
                 TGGTATTTTTCGGGGAA 
                 128 
                 CCCGACTCGAAAACC 
               
               
                   
                   
                   
                 AGTTTCG 
                   
                 TCCG 
               
               
                   
               
               
                 146 
                 MAX.chr1.110627 
                 27 
                 CGAAAGATCGATCGATT 
                 28 
                 TTATAACGACGAATTC 
               
               
                   
                 096-110627364 
                   
                 GTTCGACGG 
                   
                 CGAAA 
               
               
                   
               
               
                 146 
                 MAX.chr1.110627 
                 129 
                 TCGTTGGGTGTTCGTCG 
                 130 
                 TTACTTACTACCTCCG 
               
               
                   
                 096-110627364 
                   
                 C 
                   
                 ACTCCGC 
               
               
                   
               
               
                 151 
                 MAX.chr2.711160 
                 37 
                 ATTTTTGATTATGGGTTT 
                 38 
                 GAATTCACCTCGCCC 
               
               
                   
                 33-71116122 
                   
                 TGGTCGG 
                   
                 CTCGCT 
               
               
                   
               
               
                 151 
                 MAX.chr2.711160 
                 131 
                 TCGTATTCGGGTTTATTT 
                 132 
                 CGCGCCCTCTAACGA 
               
               
                   
                 33-71116122 
                   
                 CGTTTTTCG 
                   
                 CC 
               
               
                   
               
               
                 152 
                 MAX.chr20.21080 
                 39 
                 TAAATAAAATATTGGTTT 
                 40 
                 CGAACTAACGCTAAA 
               
               
                   
                 958-21081038 
                   
                 GTAGACGGACGCGG 
                   
                 CTCGCGCGAT 
               
               
                   
               
               
                 152 
                 MAX.chr20.21080 
                 133 
                 GTAATGTTAAATAAAATA 
                 134 
                 CGAACTAACGCTAAA 
               
               
                   
                 958-21081038 
                   
                 TTGGTTTGTAGACGGAC 
                   
                 CTCGCG 
               
               
                   
                   
                   
                 G 
                   
                   
               
               
                   
               
               
                 153 
                 MAX.chr7.155259 
                 49 
                 TTTTCGGCGGAGTCGGG 
                 50 
                 GATCTCCGCTCGCCT 
               
               
                   
                 597-155259763 
                   
                 ATTTTTTC 
                   
                 CGACG 
               
               
                   
                 (v2) 
                   
                   
                   
                   
               
               
                   
               
               
                 153 
                 MAX.chr7.155259 
                 135 
                 TTTTTGCGGTTTTCGTTC 
                 136 
                 CGAAACCGAAACCCT 
               
               
                   
                 597-155259763 
                   
                 GTTTC 
                   
                 CCCG 
               
               
                   
                 (v2) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
             
               
                 TABLE 11 
               
               
                   
               
               
                 DMR 
                   
                   
                   
               
               
                 No. 
                 Gene Annotation 
                 SEQ ID NO 
                 Probe Sequence 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 322 
                 MAX.chr10.62492680-62492822 
                 137 
                 AGGCCACGGACG 
               
               
                   
                   
                   
                 CGAATTCGGTllTAGTGTTGGA/3C6/ 
               
               
                   
               
               
                 323 
                 TMEM30B_B 
                 138 
                 CGCGCCGAGG 
               
               
                   
                   
                   
                 CGCGGTCGGTTTGTTTAT/3C6/ 
               
               
                   
               
               
                 324 
                 MAX.chr11:14926602-14926831 
                 139 
                 AGGCCACGGACG 
               
               
                   
                   
                   
                 CGTTTTTCGGATTTGGTTTTCG/3C6/ 
               
               
                   
               
               
                 325 
                 HOXA9_9650 
                 140 
                 CGCGCCGAGG 
               
               
                   
                   
                   
                 GCGCGTTTTTGCGTTT/3C6/ 
               
               
                   
               
               
                 326 
                 C10orf55_B 
                 141 
                 AGGCCACGGACG 
               
               
                   
                   
                   
                 CGCGTTTTTTTTAAATTTTTGTGAG/3C6/ 
               
               
                   
               
               
                 73 
                 MAX.chr17.73073682-73073830 
                 142 
                 CGCGCCGAGG 
               
               
                   
                   
                   
                 CGTTTCGGTTACGTTTAAGC/3C6/ 
               
               
                   
               
               
                 122 
                 TSPAN33 
                 143 
                 AGGCCACGGACG 
               
               
                   
                   
                   
                 CGCGTCGAGTTTTTTCGG/3C6/ 
               
               
                   
               
               
                 327 
                 FAM174B_C 
                 144 
                 CGCGCCGAGG 
               
               
                   
                   
                   
                 GCGACCCGTCGAATAAC/3C6/ 
               
               
                   
               
               
                 86 
                 MAX.ch_r5.60921627-60921853 
                 145 
                 AGGCCACGGACG 
               
               
                   
                   
                   
                 CGATTCGTAGTATTCGGGTTATAG/3C6/ 
               
               
                   
               
               
                 111 
                 SIX4_A 
                 146 
                 CGCGCCGAGG 
               
               
                   
                   
                   
                 CGGGATTCGCGGTTTTTAG/3C6/ 
               
               
                   
               
               
                 49 
                 KCNQ4_A 
                 147 
                 AGGCCACGGACG 
               
               
                   
                   
                   
                 GATCCGAACTCCTAAACCCG/3C6/ 
               
               
                   
               
               
                 32 
                 FOXP1 
                 148 
                 CGCGCCGAGG 
               
               
                   
                   
                   
                 CGCGCGTTTTTTTTTTTTTATAAAT/3C6/ 
               
               
                   
               
               
                 105 
                 OXT 
                 149 
                 AGGCCACGGACG 
               
               
                   
                   
                   
                 CGGTCGAGGTTTTTACGG/3C6/ 
               
               
                   
               
               
                 328 
                 C2orf82_B 
                 150 
                 CGCGCCGAGG 
               
               
                   
                   
                   
                 CGTGATCGTCGTTTTGTTG/3C6/ 
               
               
                   
               
               
                 329 
                 TAL1_B 
                 151 
                 AGGCCACGGACG 
               
               
                   
                   
                   
                 GTTCGTTTTCGATAAGCGTTTC/3C6/ 
               
               
                   
               
               
                 330 
                 BTBD19_B 
                 152 
                 CGCGCCGAGG 
               
               
                   
                   
                   
                 CGTAGGGAGTTTTCGATTCG/3C6/ 
               
               
                   
               
               
                 331 
                 MAX.chr10.22541874-22541948 
                 153 
                 AGGCCACGGACG 
               
               
                   
                   
                   
                 GCGCCTAATATCGCTAAAAAC/3C6/ 
               
               
                   
               
               
                 99 
                 MIR155HG 
                 154 
                 CGCGCCGAGG 
               
               
                   
                   
                   
                 CGAGTCGTTCGTAGAGTAAGC/3C6/ 
               
               
                   
               
               
                 85 
                 MAX.chr5.42992655-42992768 
                 155 
                 AGGCCACGGACG 
               
               
                   
                   
                   
                 CGGCGATTTGAGTGTTGT/3C6/ 
               
               
                   
               
               
                 97 
                 ME3 
                 156 
                 CGCGCCGAGG 
               
               
                   
                   
                   
                 GAACCTACGACGCCCTC/3C6/ 
               
               
                   
               
               
                 45 
                 HOPX 
                 157 
                 AGGCCACGGACG 
               
               
                   
                   
                   
                 CGCGGGTTTTTATCGATTTTC/3C6/ 
               
               
                   
               
               
                 81 
                 MAX.chr20.3229151-3229791 
                 158 
                 CGCGCCGAGG 
               
               
                   
                   
                   
                 GCGGTAGGTTGGGGT/3C6/ 
               
               
                   
               
               
                 21 
                 CLIC5 
                 159 
                 AGGCCACGGACG 
               
               
                   
                   
                   
                 GAAACCCTATACTCCCCCG/3C6/ 
               
               
                   
               
               
                 66 
                 MAX.chr13.29106812-29106917 
                 160 
                 CGCGCCGAGG 
               
               
                   
                   
                   
                 CGTTGTTTTCGCGTTTTCG/3C6/ 
               
               
                   
               
               
                 16 
                 FOXL2NB 
                 161 
                 AGGCCACGGACG 
               
               
                   
                   
                   
                 CGCGTTTTTCGTTCGATTG/3C6/ 
               
               
                   
               
               
                 136 
                 FLJ22536_A 
                 162 
                 CGCGCCGAGG 
               
               
                   
                   
                   
                 GAAACGAACTAAAACTACTCGATCC/3C6/ 
               
               
                   
               
               
                 146 
                 MAX.chr1.110627096- 
                 163 
                 AGGCCACGGACG 
               
               
                   
                 110627364 
                   
                 CGCGGTTAGGTTTGCG/3C6/ 
               
               
                   
               
               
                 146 
                 MAX.chr1.110627096- 
                 164 
                 CGCGCCGAGG 
               
               
                   
                 110627364 
                   
                 CGCGGTTAGGTTTGCG/3C6/ 
               
               
                   
               
               
                 151 
                 MAX.chr2.71116033-71116122 
                 165 
                 CGCGCCGAGG 
               
               
                   
                   
                   
                 CGCGAAACGAAAACACCT/3C6/ 
               
               
                   
               
               
                 152 
                 MAX.chr20.21080958-21081038 
                 166 
                 AGGCCACGGACG 
               
               
                   
                   
                   
                 GCGATTCCCAAAACCCG/3C6/ 
               
               
                   
               
               
                 153 
                 MAX.chr7.155259597- 
                 167 
                 CGCGCCGAGG 
               
               
                   
                 155259763 
                   
                 CGGCGGTTTTCGAAGC/3C6/ 
               
               
                   
               
            
           
         
       
     
     INCORPORATION BY REFERENCE 
     The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes. 
     EQUIVALENTS 
     The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.