Patent Publication Number: US-11655497-B2

Title: Method of amplifying a target nucleic acid

Description:
BACKGROUND OF THE INVENTION 
     Human genetic mutations whether it is de novo or somatic are critical information to understand human genetic disease (Ku, C. S. et al, A new era in the discovery of de novo mutations underlying human genetic disease, Hum Genomics 6, 27, 2012), cancer biology (Helleday, T. et al, Mechanisms underlying mutational signatures in human cancers, Nat Rev Genet 15, 585-598, 2014) and potential anticancer therapies. de novo mutation has long been known to cause genetic disease and it also plays an important role in rare and common forms of neurodevelopmental diseases, including intellectual disability, autism and schizophrenia (Veltman, J. A. et al, De novo mutations in human genetic disease, Nat Rev Genet 13, 565-575, 2012). Somatic mutation in cancer genome has been extensively studied and believed to hold the key to understand cancer origin, risk and potential biomarker discovery for therapeutic use. Detection of those genetic mutations is critical for diagnosis of disease and patient treatment. 
     Studies of de novo or somatic mutations in the human genome have been very challenging in the past because of genomic sequencing technology limitations. However, the development of high-throughput next-generation sequencing (NGS) technologies has greatly facilitated the study of such mutations. Whole-genome sequencing (WGS) and whole-exome sequencing (WES) can now be performed on parent offspring trios to identify de novo point mutations in the entire genome or within protein-coding regions, respectively. 
     WGS and WES are great tools for genetic mutation study, but they are still cost prohibitive for routine clinical use. In some cases, if only a set of genetic mutations are known to be related with certain disease or particular drug response, it would be efficient and cost effective to do genetic analysis for those genes. In order to do a limited resequencing of panel of genes, those genes need to be captured before carrying out NGS. The capture process could be achieved using either hybridization or amplicon approach. For hybridization capture approach, gDNA was first physically fragmented or enzymatically digested, then synthetic oligonucleotides are hybridized to regions of interest in solution to capture the intended sequences. For amplicon based approach, the intended regions are directly captured by amplification of PCR primers. Hybridization capture approach is scalable to large number of genes, but hybridization step usually takes overnight and the total process takes multiple days. It also requires at least 1 to 2 μg of gDNA material input. Amplicon based approach takes less time and only require 10 to 50 ng gDNA input, so it is suitable if quantities of DNA input from clinical samples are limited. However, multiplex PCR primers also generate nonspecific amplification products especially when the number of PCR primers increase. In fact, majority of PCR products are nonspecific amplicons when the number of primers approaches hundreds. Therefore, amplicon based approach usually uses an enzyme digestion step to reduce nonspecific amplification product followed by additional ligation step or use a multiple steps of cleaning up to reduce those nonspecific products. Those nonspecific amplification products not only require multiple steps during sequencing library generation but also can introduce sequencing data errors. 
     Recently detection of low frequency mutation has been a rapidly growing area of interest because of its important applications in basic and clinical research. One kind of rare mutations, circulating cell-free DNA (cfDNA) from human plasma are used for prenatal screening (Chiu, R. W. et al, Noninvasive prenatal diagnosis empowered by high-throughput sequencing, Prenat Diagn 32, 401-406, 2012), while circulating tumor DNA (ctDNA) has been confirmed to contain the hallmark mutations of cancerous cells. ctDNA has the potential to be a novel, non-invasive biomarker that promotes early cancer detection at a surgically curable stage, reduces the necessity of repeat tissue biopsies, and detects the early relapse of the disease, thereby increasing the efficacy of targeted therapy. For cancers that are often detected at a late stage, including lung, pancreatic, and ovarian etc., a high-sensitivity ctDNA assay could be used as an important screening test to detect typically terminal metastatic stage cancer at an earlier, potentially curable stage. With continuous ctDNA monitoring from patient blood, change of ctDNA composition and quantitation could be used to monitor cancer progression in real time, improving patient safety and eliminating the cost related to repeat tissue biopsies. 
     Unfortunately, detection of ctDNA remains challenging by its presence in relatively low quantities especially in early-stage cancer patients. There are several available techniques developed so far to detect ctDNA including BEAMing, digital PCR, and next generation sequencing. All those methods can detect low frequency mutations by assessing individual molecules one-by-one. NGS has the advantage over traditional methods in that large amount of sequencing information can be obtained easily in an automated fashion. However, NGS cannot generally be used to detect rare mutations because of its high error rate associated with NGS library generation and the sequencing process. Some of these errors presumably result from mutations introduced during template preparation, during the pre-amplification steps required for library preparation and during further solid-phase amplification on the instrument itself. Other errors are due to base mis-incorporation during sequencing and base-calling errors. 
     Therefore, there remains a continuing need for a novel approach to eliminate nonspecific amplification products during multiplex PCR reaction so that the sequencing library could be directly generated without additional digestion and ligation steps, and a novel approach to reduce error rate so that rare mutation could be reliably detected using current NGS instrument. 
     BRIEF SUMMARY OF THE INVENTION 
     One aspect of the present disclosure provides a method of amplifying a target nucleic acid, wherein the method comprises: (a) providing a reaction mixture comprising: (i) a nucleic acid sample comprising or suspected of comprising the target nucleic acid, (ii) at least 20 different types of primer pairs, wherein at least one primer of each type of primer pairs is complementary to a portion of the target nucleic acid, and each primer pair has at least one blocking primer comprising a blocking group capable of blocking polymerase extension, (iii) nucleic acid polymerase, and (iv) de-blocking agent capable of enabling polymerization of the target nucleic acid by said nucleic acid polymerase using the blocking primers; and (b) incubating the reaction mixture under a condition for amplification of the target nucleic acid. 
     In some embodiments, the blocking group is at or near 3′ terminal of each blocking primer. In some embodiments, the blocking group is 2′, 3′-dideoxynucleotide, ribonucleotide residue, 2′, 3′SH nucleotide, or 2′-O—PO 3  nucleotide. 
     In some embodiments, the blocking primer is complementary to a portion of the target nucleic acid. In some embodiments, the blocking primer is further modified to decrease the amplification of undesired nucleic acid. In some embodiments, the modification is introduction of at least one mismatched nucleotide in the primer. In some embodiments, the mismatched nucleotide is 2-18 bp away from the nucleotide with the blocking group. In some embodiments, wherein the mismatched nucleotide is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 bp away from the nucleotide with the blocking group. In some embodiments, the mismatched nucleotide base is located on the 5′ side of the nucleotide with the blocking group. In some embodiments, the modification is a modification to decrease the Tm between the blocking primer and the undesired nucleic acid. In some embodiments, the modification is a modification to increase the Tm between the blocking primer and the target nucleic acid. In some embodiments, wherein the modification is a modification to form an extra bridge connecting the 2′ oxygen and 4′ carbon of at least one nucleotide of the blocking primer. 
     In some embodiments, there are no more than 20 complementary nucleotide pairings and no more than 50% sequence complementarity between any two primers. In some embodiments, there are no more than 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 complementary nucleotide pairings between any two primers. 
     In some embodiments, the reaction mixture comprises at least 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 400, 500, 600, 700, 800, 900 or 1000 different types of primer pairs. 
     In some embodiments, each of the primers is 8 to 100 nucleotides in length. 
     In some embodiments, the different types of primer pairs can complementarily bind to different target nucleic acids or different sequences in the same target nucleic acid. 
     In some embodiments, wherein the de-blocking agent is CS5 DNA polymerase with the mutations selected from G46E, L329A, Q601R, D640G, I669F, S671F, E678G or the combination of such mutations, ampliTaq or KlenTaq polymerase with F667Y mutation, pyrophosphate or RNase H2. 
     In some embodiments, the target nucleic acid is single stranded or double stranded DNA. 
     In some embodiments, the target nucleic acid is double stranded DNA ligated with single or double adaptor tags or single stranded DNA ligated with single adaptor tag. 
     In some embodiments, the reaction mixture further comprises at least one primer complementary in whole or in part with the adaptor tag. 
     In some embodiments, the target nucleic acid is double stranded DNA comprising single or double molecular index tag or single stranded DNA comprising single molecular index tag. In some embodiments, the molecular index tag comprises unique identifier nucleic acid sequence and an adaptor tag. 
     In some embodiments, the primers have common tailing sequence at or near 5′ terminal of the primers. In some embodiments, the common tailing sequence can be used as molecular index tag, sample index tag or adaptor tag or combinations of all three tags. 
     In some embodiments, the reaction mixture further comprises high fidelity polymerase. In some embodiments, the high fidelity polymerase is PFU DNA Polymerase. 
     In some embodiments, the step (b) “incubating the reaction mixture under a condition for amplification of the target nucleic acid” comprises the steps of denaturing the target nucleic acid; annealing the primers with the target nucleic acid to allow the formation of a nucleic acid-primer hybrid; and incubating the nucleic acid-primer hybrid to allow the nucleic acid polymerase to amplify the target nucleic acid. 
     In some embodiments, the formation of a nucleic acid-primer hybrid results in de-blocking the block group in the primer through de-blocking agent. 
     In some embodiments, the steps of “denaturing the target nucleic acid; annealing the primers with the target nucleic acid to allow the formation of a nucleic acid-primer hybrid; and incubating the nucleic acid-primer hybrid to allow the nucleic acid polymerase to amplify the target nucleic acid” is repeated at least 1 time, 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times or 50 times. In some embodiments, the step (b) is repeated from about 20 times to about 50 times. 
     In some embodiments, the nucleic acid sample comprises the target nucleic acid. In some embodiments, the target nucleic acids in the nucleic acid sample is no more than 1 copy, 2 copies, 5 copies, 8 copies, 10 copies, 20 copies, 30 copies, 50 copies, 80 copies or 100 copies. In some embodiments, the molar percentage of target nucleic acid in the nucleic acid sample is less than 50%, 20%, 10%, 5%, 3%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, or 0.001%. 
     In some embodiments, the nucleic acid other than the target nucleic acid is not amplified in step (b) substantially. In some embodiments, the molar percentage of undesired nucleic acid in the reaction products obtained from step (b) is less than 20%, 15%, 10%, 5%, 3%, 2% or 1%. 
     In some embodiments, the method is used for selective enrichment of mutant nucleic acid in a sample comprising wildtype nucleic acid. In some embodiments, wherein at least one blocking primer is complementary to the mutant nucleic acid at the mutant residues and the nucleotide of the blocking primer corresponding to a mutant residue has the blocking group. 
     Another aspect of the present disclosure provides a method of sequencing a target nucleic acid, wherein the method comprises: (a) providing a reaction mixture comprising: (i) a nucleic acid sample comprising or suspected of comprising the target nucleic acid, (ii) at least 20 different types of primer pairs, wherein at least one primer of each type of primer pairs is complementary to a portion of the target nucleic acid, and each primer pair has at least one blocking primer comprising a blocking group capable of blocking polymerase extension, (iii) nucleic acid polymerase, and (iv) de-blocking agent capable of enabling polymerization of the target nucleic acid by said nucleic acid polymerase using the blocking primers; (b) incubating the reaction mixture under a condition for amplification of the target nucleic acid; and (c) determining the sequence of the reaction products obtained from step (b). 
     In some embodiments, the method is used for sequencing by capillary electrophoresis, PCR or high throughput sequencing. In some embodiments, the blocking primer is further modified to decrease the amplification of undesired nucleic acid. 
     In some embodiments, the reaction mixture further comprises high fidelity polymerase. 
     Yet another aspect of the present disclosure provides a method of sequencing a target nucleic acid, wherein the method comprises: (a) providing a reaction mixture comprising: (i) a nucleic acid sample comprising or suspected of comprising the target nucleic acid, (ii) at least 20 different types of primer pairs, wherein at least one primer of each type of primer pairs is complementary to a portion of the target nucleic acid, and each primer pair has at least one blocking primer comprising a blocking group capable of blocking polymerase extension, (iii) nucleic acid polymerase, and (iv) de-blocking agent capable of enabling polymerization of the target nucleic acid by said nucleic acid polymerase using the blocking primers; (b) incubating the reaction mixture under a condition for amplification of the target nucleic acid; (c) adding adaptor tag, molecular index tag and/or sample index tag to the reaction products obtained from step (b); and (d) determining the sequence of the reaction products obtained from step (c). 
     In some embodiments, the method is used for sequencing by capillary electrophoresis, PCR or high throughput sequencing. 
     In some embodiments, wherein the blocking primer is modified to decrease the amplification of undesired nucleic acid. 
     In some embodiments, wherein the reaction mixture further comprises high fidelity polymerase. 
     Yet another aspect of the present disclosure provides a kit for amplifying a target nucleic acid, wherein the kit comprises: (i) at least 20 different types of primer pairs, wherein at least one primer of each type of primer pairs is complementary to a portion of the target nucleic acid, and each primer pair has at least one blocking primer comprising a blocking group capable of blocking polymerase extension, (ii) nucleic acid polymerase, and (iii) de-blocking agent capable of enabling polymerization of the target nucleic acid by said nucleic acid polymerase using the blocking primer. 
     In some embodiments, the blocking primer is modified to decrease the amplification of undesired nucleic acid. 
     In some embodiments, the reaction mixture further comprises high fidelity polymerase. 
     Yet another aspect of the present disclosure provides a method of amplifying a target nucleic acid, wherein the method comprises: (a) providing a reaction mixture comprising: (i) a nucleic acid sample comprising or suspected of comprising the target nucleic acid, (ii) at least one type of primers that is complementary to a portion of the target nucleic acid, and each type of primers has at least one blocking primer comprising a blocking group capable of blocking polymerase extension, wherein the blocking primer is modified to decrease the amplification of undesired nucleic acid, (iii) nucleic acid polymerase, and (iv) de-blocking agent capable of enabling polymerization of the target nucleic acid by said nucleic acid polymerase using the blocking primer; and (b) incubating the reaction mixture under a condition for amplification of the target nucleic acid. 
     In some embodiments, the modification is introduction of at least one mismatched nucleotide in the primer. 
     In some embodiments, the mismatched nucleotide is 2-18 bp away from the nucleotide with the blocking group. 
     In some embodiments, the mismatched nucleotide is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 bp away from the nucleotide with blocking group. 
     In some embodiments, the mismatched nucleotide is located on the 5′ side of the blocking group. 
     In some embodiments, the modification is a modification to decrease the affinity between the blocking primer and the target nucleic acid. 
     In some embodiments, the modification is a modification to form an extra bridge connecting the 2′ oxygen and 4′ carbon of at least one nucleotide of the blocking primer. 
     In some embodiments, the method is used for selective enrichment of mutant nucleic acid in a sample comprising wild type nucleic acid. 
     In some embodiments, a blocking primer is complementary to a portion of the target nucleic acid. In some embodiments, the blocking primer is complementary to the mutant nucleic acid at the mutant residue and the nucleotide of the blocking primer corresponding to a mutant residue has the blocking group. 
     Yet another aspect of the present disclosure provides a kit for amplifying a target nucleic acid, wherein the kit comprises: (i) at least one type of primers that is complementary to a portion of the target nucleic acid, and each type of primers have at least one blocking primer comprising a blocking group capable of blocking polymerase extension, wherein the blocking primer is modified to decrease the amplification of undesired nucleic acid, (ii) nucleic acid polymerase, and (iii) de-blocking agent capable of enabling polymerization of the target nucleic acid by said nucleic acid polymerase using the blocking primer. 
     In some embodiments, the blocking primer is modified to decrease the affinity between the blocking primer and the target nucleic acid. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG.  1   : NGS library construction for genomic DNA by multiplex PCR. 
         FIG.  2   : NGS library construction for fragmented DNA by multiplex PCR. 
         FIG.  3   : NGS library construction for fragmented DNA with single stranded molecular index tag by multiplex PCR. 
         FIG.  4   : NGS library construction for fragmented DNA with double stranded molecular index tags by multiplex PCR. 
         FIG.  5   : Selectively amplification of mutant sequence in genomic DNA by multiplex PCR. 
         FIG.  6   : Mutant enriched NGS library construction for fragmented DNA by multiplex PCR. 
         FIG.  7   : Mutant enriched NGS library construction for fragmented DNA with single stranded molecular index tag by multiplex PCR. 
         FIG.  8   : NGS library construction for fragmented DNA with double stranded molecular index tags by multiplex PCR. 
         FIG.  9   . The normalized reads per amplicon in a 196-plex reaction on a genomic DNA sample across six individual reactions followed by sequencing run on a MiSeq sequencer in Example 1. 
         FIG.  10   . The normalized reads per amplicon v.s. amplicon GC percentage in a 196-plex reaction on a genomic DNA sample across six individual reactions followed by sequencing run on a MiSeq sequencer in Example 1. 
         FIG.  11   . General working flow for multiplex PCR reaction assay design and NGS data analysis. 
         FIG.  12   . Electropherogram of selectively enriched different mutant nucleic acids after multiplex PCR reaction in Example 2. 
         FIG.  13   . Electropherogram of selectively enriched mutant nucleic acid after multiplex PCR reaction in Example 3. 
         FIG.  14   . Electropherogram of selectively enriched mutant nucleic acid after multiplex PCR reaction in Example 4. 
         FIG.  15   . The sketch of multiplex PCR and the construction of library in Example 1. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     One aspect of the present disclosure provides a method of amplifying a target nucleic acid, wherein the method comprises: (a) providing a reaction mixture; and (b) incubating the reaction mixture under a condition for amplification of the target nucleic acid. 
     Providing a Reaction Mixture 
     In some embodiments, a reaction mixture for detecting a target nucleic acid of the present disclosure comprises: (i) a nucleic acid sample comprising or suspected of comprising the target nucleic acid, (ii) at least 20 different types of primer pairs, wherein at least one primer of each type of primer pairs is complementary to a portion of the target nucleic acid, and each primer pair has at least one blocking primer comprising a blocking group capable of blocking polymerase extension, (iii) nucleic acid polymerase, and (iv) de-blocking agent capable of enabling polymerization of the target nucleic acid by said nucleic acid polymerase using the blocking primers. 
     Nucleic Acid Sample 
     The term “nucleic acid” as used in the present disclosure refers to a biological polymer of nucleotide bases, and may include but is not limited to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), micro RNA (miRNA), and peptide nucleic acid (PNA), modified oligonucleotides (e.g., oligonucleotides comprising nucleotides that are not conventional to biological RNA or DNA, such as 2′-O-methylated oligonucleotides), and the like. The nucleotides of the present disclosure can be natural or unnatural, substituted or unsubstituted, modified or unmodified. The nucleotides can be linked by phosphodiester bonds, or by phosphorothioate linkages, methylphosphonate linkages, boranophosphate linkages, or the like. The polynucleotides can additionally comprise non-nucleotide elements such as labels, quenchers, blocking groups, or the like. The nucleic acid can be, e.g., single-stranded or double-stranded. 
     The term “DNA” as used in the present disclosure refers to deoxyribonucleic acid, a long chain polymer biological macromolecule which forms genetic instructions. The subunit of DNA is nucleotide. Each nucleotide in DNA consists of a nitrogenous base, a five-carbon sugar (2-deoxyribose) and phosphate groups. Neighboring nucleotides are linked via diester bonds formed by deoxyribose and phosphoric acid, thereby forming a long chain framework. Generally, there are four types of nitrogenous bases in DNA nucleotides, namely adenine (A), guanine (G), and cytosine (C), thymine (T). The bases on the two DNA long chains pair via hydrogen bonds, wherein adenine (A) pairs with thymine (T), and guanine (G) pairs with cytosine (C). 
     The term “nucleic acid sample” as used in the present disclosure refers to any sample containing nucleic acid, including but not limited to cells, tissues, and body fluids, etc. In some embodiments, the nucleic acid sample is a tissue, e.g., biopsy tissue or paraffin embedded tissue. In some embodiments, the nucleic acid sample is bacteria or animal or plant cells. In some other embodiments, the nucleic acid sample is a body fluid, e.g., blood, plasma, serum, saliva, amniocentesis fluid, pleural effusion, seroperitoneum, etc. In some embodiments, the nucleic acid sample is blood, serum or plasma. 
     In some embodiments, the nucleic acid sample comprises or is suspected of comprising the target nucleic acid. 
     The term “target nucleic acid” or “target region” as used in the present disclosure refers to any region or sequence of a nucleic acid which is to be amplified intentionally. 
     In some specific embodiments, the target nucleic acid is DNA, RNA or a hybrid or a mixture thereof. In some specific embodiments, the target nucleic acid is genomic DNA. In some specific embodiments, the target nucleic acid is cell-free DNA (cfDNA). In some specific embodiments, the target nucleic acid is circulating tumor DNA (ctDNA). 
     “Cell-free DNA” as used in the present disclosure refers to DNA released from cells and found in circulatory system (e.g., blood), the source of which is generally believed to be genomic DNA released during apoptosis. 
     “Circulating tumor DNA” as used in the present disclosure refers to the cell-free DNA originated from tumor cells. In human body, a tumor cell may release its genomic DNA into the blood due to causes such as apoptosis and immune responses. Since a normal cell may also release its genomic DNA into the blood, circulating tumor DNA usually constitutes only a very small part of cell-free DNA. 
     In some embodiments, the target nucleic acid is single stranded or double stranded DNA. In some embodiments, the target nucleic acid is the whole or a portion of one or more genes selected from ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53 and VHL. 
     In some embodiments, the amount of target nucleic acid in the nucleic acid sample is no more than 1 copy, 2 copies, 3 copy, 4 copies, 5 copies, 6 copies, 7 copies, 8 copies, 9 copies, 10 copies, 12 copies, 15 copies, 18 copies, 20 copies, 30 copies, 50 copies, 80 copies or 100 copies. In some embodiments, the molar percentage (molar/molar) of target nucleic acid in the nucleic acid sample is less than 50%, 20%, 10%, 8%, 6%, 5%, 3%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, or 0.001%. In some embodiments, the ratio of molar of target nucleic acid and the molar of un-target nucleic acid in the nucleic acid sample is less than 50%, 20%, 10%, 8%, 6%, 5%, 3%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, or 0.001%. 
     In some embodiments, the target nucleic acid is DNA fragment. In some embodiments, the size of the target nucleic acid is 0.01-5 kb, 0.1-5 kb, 0.1-1 kb, 1-2 kb, 2-3 kb, 3-4 kb, 4-5 kb, 0.2-0.4 kb, 0.5-1 kb, 0.1-0.5 kb, 0.01-0.5 kb, 0.01-0.4 kb, 0.01-0.3 kb, 0.01-0.25 kb, 0.02-0.25 kb, 0.05-0.3 kb or 0.05-0.25 kb. The DNA fragment can be obtained through common technology in the art (e.g., physical breaking, cleavage using specific restriction endonuclease, etc.). 
     In some embodiments, the target nucleic acid is double stranded DNA ligated with single or double adaptor tags or single stranded DNA ligated with single adaptor tag. 
     The term “adaptor tag” as used in the present disclosure refers to a specific DNA sequence attached to one or two ends of a nucleic acid (single stranded or double stranded) according to needs, and the length of the adaptor is usually within 5-50 bp. The adaptor tag can be used to facilitate amplifying the target nucleic acid and/or sequencing the amplified target nucleic acid. In some embodiments, the adaptor tag is used to facilitate the ligation of tags for sequencing (e.g., the ligation of P5 and P7 tag for Illumina MiSeq sequencer). In some embodiments, the adaptor tag is attached to only one end of a single stranded nucleic acid at 3′ terminal or 5′ terminal. In some embodiments, the adaptor tag is attached to two ends of a single stranded nucleic acid. In some embodiments, one adaptor tag is attached to each strand in double stranded nucleic acid at 3′ terminal or 5′ terminal. For example, one adaptor tag is attached to one strand in double stranded nucleic acid at its 3′ terminal and one adaptor tag is attached to the other strand in double stranded nucleic acid at its 5′ terminal, and the two adaptor tags are identical or complementary to each other. In some embodiments, two adaptor tags are attached to two ends of each strand in double stranded nucleic acid. 
     The adaptor tag can be attached to the nucleic acid through common technologies in the art. In some embodiments, where the target nucleic acid is double stranded DNA, the adaptor tag can be attached to the nucleic acid through the following steps: (a) providing an adaptor ligation nucleic acid designed to contain sequences to ligate with an end of one strand of the DNA (for example, the adaptor ligation nucleic acid contains a hybridization complementary region, or a random hybridization short sequence, e.g., poly-T); (b) hybridization of the adaptor ligation nucleic acid and the strand of the DNA; and (c) adding polymerase (e.g., reverse transcriptase) after the hybridization to extend the adaptor ligation nucleic acid, thereby the adaptor tag is ligated to the end of the target DNA fragment. For attaching another adaptor to the other end of the same strand or to the other strand of the DNA, an adaptor ligation nucleic acid can be designed according to the needs and steps (b)-(c) can be repeated. In some other embodiments, where the DNA fragment is double stranded and the end of the DNA fragment is a sticky end, the adaptor tag can be attached to the nucleic acid through the following steps: (a) designing the adaptor ligation nucleic acid to contain sequences to ligate with the sticky end; (b) complementarily annealing the adaptor ligation nucleic acid with the sticky end; and (c) ligating the adaptor ligation nucleic acid to the double stranded of the target DNA using a ligase, thereby achieving the purpose of attaching the adapter to the end of the DNA fragment. 
     In some embodiments, the target nucleic acid is double stranded DNA comprising single or double molecular index tags or single stranded DNA comprising single molecular index tag. In some embodiments, the molecular index tag comprises unique identifier nucleic acid sequence and an adaptor tag. In some embodiments, the adaptor tag is at one end of the target nucleic acid. 
     The term “molecular index tag” as used in the present disclosure refers to a nucleic acid sequence used as a tag, which can be ligated to or existing at the 5′ end, the 3′ end or both ends of a DNA fragment. In DNA sequencing, especially in high throughout sequencing technology, a molecular index tag therein is used to mark particular DNA molecule. After amplification and sequencing, the count of the molecular index sequence therein is used to mark particular DNA molecule and can be the basis for determining the quantity of expression of the marked gene, or be used to trace the information of the amplified DNA molecules from the same original molecules and thereby correcting the random errors of DNA sequences during amplification and sequencing. 
     In some embodiments, the molecular index tag is exogenous, which is attached to the target nucleic acid through PCR (e.g., as described in MoCloskey M. L. et al, Encoding PCR products with batch-stamps and barcodes. Biochem Genet 45:761-767, 2014 or Parameswaran P, et al., A pyrosequencing-tailored nucleotide barcode design unveils opportunities for large-scale sample multiplexing. Nucleic Acids Res 35:e130, 2017) or ligation (e.g., as described in Craig D W, et al., Identification of genetic variants using bar-coded multiplexed sequencing. Nat Methods 5:887-893, 2008 or Miner B E, et al., Molecular barcodes detect redundancy and contamination in hairpin-bisulfite PCR. Nucleic Acids Res 32:e135, 2004). In some embodiments, the molecular index tag or the unique identifier nucleic acid sequence therein can be a random sequence (i.e., formed with randomly arranged A/T/C/G). 
     In some embodiments, the molecular index tag or the unique identifier nucleic acid sequence therein is endogenous, which are the sequences of the two ends of randomly sheared fragment. 
     More information for molecular index tag can be found in U.S. 20140227705 and U.S. 20150044687. 
     Primer 
     The term “primer” as used in the present disclosure refers to an oligonucleotide that is capable of acting as a point of initiation of synthesis when placed under conditions in which primer extension is initiated. Primer may comprise natural ribonucleic acid, deoxyribonucleic acid, or other forms of natural nucleic acid. Primer may also comprise un-natural nucleic acid (e.g., LNA, ZNA etc.). 
     Primers may be prepared using any suitable method, such as, for example, the phosphotriester and phosphodiester methods or automated embodiments thereof. In one such automated embodiment, diethylophosphoramidites are used as starting materials and may be synthesized as described by Beaucage et al., Tetrahedron Letters, 22:1859-1862 (1981). One method for synthesizing primer on a modified solid support is described in U.S. Pat. No. 4,458,006. It is also possible to use a primer which has been isolated from a biological source, such as a restriction endonuclease digest. In some embodiments, the primer with blocking nucleotide at the 3′ end, can be synthesized with terminal transferase (Gibco BRL) (Nuc Aci Res 2002, 30(2)). 
     The term “primer pair” as used in the present disclosure refers to a pair of primers consisting of a forward primer and a reverse primer which complement with a portion of a sequence to be amplified, respectively, wherein the forward primer defines a point of initiation of the amplified sequence and the reverse primer defines a point of termination of the amplified sequence. The term “complimentary”, when it is used to describe the relationship between primer and the sequence to be amplified, refers to that the primer is complimentary to the sequence to be amplified or is complimentary to a complementary sequence of the sequence to be amplified. 
     The pair of primers can be designed based on the sequence of the target nucleic acid. In some embodiments, at least one primer of each type of primer pairs is complementary to a portion of the target nucleic acid. In some embodiments, when the target sequence (assuming it is a double stranded DNA) has an adaptor tag, one primer of a primer pair may be complementary to a portion of the target sequence (on one strand) and the other primer may be complementary to the adaptor tag (on the other strand). 
     In some embodiments, each primer pair has at least one blocking primer comprising a blocking group capable of blocking polymerase extension. In some embodiments, both primers in each primer pair are blocking primers comprising a blocking group capable of blocking polymerase extension. 
     The term “blocking primer” as used in the present disclosure refers to a primer having a blocking group. 
     The term “blocking group” as used in the present disclosure refers to any chemical group covalently linked in a nucleic acid chain and capable of blocking polymerase extension. In some embodiments, the nucleotide with blocking group is a modified nucleotide at or near the 3′ terminal of each blocking primer. In some embodiments, the nucleotide with blocking group is no more than 6 bp, 5 bp, 4 bp, 3 bp, 2 bp or 1 bp away from the 3′ terminal of each blocking primer. In some embodiments, when the method of the present disclosure is used for selective enrichment of mutant nucleic acid in a sample comprising wildtype nucleic acid, the blocking group is at the nucleotide that is complementary with the corresponding mutated nucleotide of the mutant nucleic acid but is not complementary with the corresponding nucleotide of wildtype nucleic acid. 
     In some embodiments, the blocking group is 2′, 3′-dideoxynucleotide, ribonucleotide residue, 2′, 3′SH nucleotide, or 2′-O—PO 3  nucleotide. When the blocking group is a ribonucleotide residue, the blocking primer is a primer that has one ribonucleotide residue and other residues are all deoxyribonucleotide residues. 
     More information for blocking group and blocking primer can be found in U.S. Pat. Nos. 9,133,491, 6,534,269 and Joseph R. D. et al., RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers, BMC Biotechnology 11:80, 2011. 
     In some embodiments, the blocking primer is complementary to a portion of the target nucleic acid. 
     In some embodiments, the primers are 5 to 100 nucleotides in length. In some embodiments, the primers are at least 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 or 40 nucleotides in length. In some embodiments, the primers are no more than 100, 90, 80, 70, 60, 50, 40, 35, 30, 25 or 20 nucleotides in length. 
     In some embodiments, a primer comprises a complementary region that is complementary to the target sequence and a common tailing sequence at or near the 5′ terminal of the primer. In some embodiments, the common tailing sequence can be used as molecular index tag, adaptor tag or sample index tag or combinations of all the three tags. 
     The term “sample index tag” as used in the present disclosure refers to a series of unique nucleotides (i.e., each sample index tag is unique), and can be used to allow for multiplexing of samples such that each sample can be identified based on its sample index tag. In some embodiments, there is a unique sample index tag for each sample in a set of samples, and the samples are pooled during sequencing. For example, if twelve samples are pooled into a single sequencing reaction, there are at least twelve unique sample index tags such that each sample is labeled uniquely. 
     In some embodiments, the blocking primer is modified so as to further decrease the amplification of undesired nucleic acid. 
     In some embodiments, the modification is introduction of at least one mismatched nucleotide in the primer. In some embodiments, the mismatched nucleotide base is located on the 5′ side of the nucleotide with the blocking group. 
     The term “mismatched nucleotide” as used in the present disclosure refers to a nucleotide of a first nucleic acid (e.g., primer) that is not capable of pairing with a nucleotide at a corresponding position of a second nucleic acid (e.g., target nucleic acid), when the first and second nucleic acids are aligned. 
     The preferred or accepted location of the mismatched nucleotide can be determined through conventional technologies. For example, the mismatched nucleotides are introduced into different locations in the blocking primer, and those blocking primers are used for amplifying a target nucleic acid separately, and then the preferred or accepted location of the mismatched nucleotide for the target nucleic acid can be determined based on the results of amplification (e.g., the location decreasing the amplification of undesired nucleic acid or false positive results is preferred or accepted location). The location of the mismatched nucleotide may change along with the change of the target nucleic acid or the structure of the blocking primer. In some embodiments, the mismatched nucleotide is 2-18 bp away from the nucleotide with blocking group. In some embodiments, the mismatched nucleotide is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 bp away from the nucleotide with blocking group. In some embodiments, the mismatched nucleotide is no less than 2, 3, 4, 5, 6, 7, 8, 9 or 10 bp away from the nucleotide with blocking group. In some embodiments, the mismatched nucleotide is no more than 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 bp away from the nucleotide with blocking group. 
     In some embodiments, the modification is a modification to increase the melting temperature (Tm) between the blocking primer and the target nucleic. In some embodiments, the modification is a modification to decrease the melting temperature (Tm) between the blocking primer and the undesired nucleic acid which may be the wildtype nucleic acid in a method for selective enrichment of mutant nucleic acid in a sample. In some embodiments, wherein the modification is a modification to form an extra bridge connecting the 2′ oxygen and 4′ carbon of at least one nucleotide of the blocking primer, such as locked nucleic acid (LNA), see, e.g., Karkare S et al., Promising nucleic acid analogs and mimics: characteristic features and applications of PNA, LNA, and morpholino. Appl Microbiol Biotechnol 71(5):575-586, 2006 and VesterB et al., LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA. Biochemistry 43(42):13233-13241, 2004. 
     In some embodiments, the reaction mixture comprises at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 400, 500, 600, 700, 800, 900 or 1000 different types of primer pairs. In some embodiments, the different types of pairs of the primers are complementary to different target nucleic acid fragments or are complementary to different sequences in the same target nucleic acid fragment. 
     The inventors of the present disclosure conducted a simulation experiment to evaluate the probability of forming primer pairs between primers with certain lengths generated randomly. The inventors randomly generated from 10 to 490 primer pairs in length of 20 bp to form different primer pools and, for each pool, checked primer dimer formation between any one primer and other primer in the same pool. It can be seen that the probability to form primer dimer (e.g., resulting from complementarily between different primers) is increased along with the increasing numbers of primers. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Relationship between the number of the primers 
               
               
                 and the dimer length. 
               
            
           
           
               
               
               
            
               
                   
                 Number of 
                 Dimer Length 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 Primer Pairs 
                 4 bp 
                 5 bp 
                 6 bp 
                 7 bp 
                 8 bp 
               
               
                   
                   
               
               
                   
                  10 
                 100% 
                  90% 
                  20% 
                  0% 
                  0% 
               
               
                   
                  30 
                 100% 
                 100% 
                  67% 
                  7% 
                  7% 
               
               
                   
                  50 
                 100% 
                 100% 
                  88% 
                 22% 
                  0% 
               
               
                   
                  70 
                 100% 
                 100% 
                  94% 
                 50% 
                 20% 
               
               
                   
                  90 
                 100% 
                 100% 
                  96% 
                 48% 
                  9% 
               
               
                   
                 110 
                 100% 
                 100% 
                 100% 
                 72% 
                 13% 
               
               
                   
                 130 
                 100% 
                 100% 
                  99% 
                 61% 
                 23% 
               
               
                   
                 150 
                 100% 
                 100% 
                  99% 
                 72% 
                 23% 
               
               
                   
                 170 
                 100% 
                 100% 
                 100% 
                 79% 
                 29% 
               
               
                   
                 190 
                 100% 
                 100% 
                  99% 
                 77% 
                 32% 
               
               
                   
                 210 
                 100% 
                 100% 
                 100% 
                 82% 
                 31% 
               
               
                   
                 230 
                 100% 
                 100% 
                 100% 
                 83% 
                 34% 
               
               
                   
                 250 
                 100% 
                 100% 
                 100% 
                 87% 
                 34% 
               
               
                   
                 270 
                 100% 
                 100% 
                 100% 
                 91% 
                 35% 
               
               
                   
                 290 
                 100% 
                 100% 
                 100% 
                 90% 
                 39% 
               
               
                   
                 310 
                 100% 
                 100% 
                 100% 
                 96% 
                 49% 
               
               
                   
                 330 
                 100% 
                 100% 
                 100% 
                 96% 
                 47% 
               
               
                   
                 350 
                 100% 
                 100% 
                 100% 
                 96% 
                 51% 
               
               
                   
                 370 
                 100% 
                 100% 
                 100% 
                 95% 
                 48% 
               
               
                   
                 390 
                 100% 
                 100% 
                 100% 
                 96% 
                 53% 
               
               
                   
                 410 
                 100% 
                 100% 
                 100% 
                 97% 
                 52% 
               
               
                   
                 430 
                 100% 
                 100% 
                 100% 
                 97% 
                 54% 
               
               
                   
                 450 
                 100% 
                 100% 
                 100% 
                 97% 
                 54% 
               
               
                   
                 470 
                 100% 
                 100% 
                 100% 
                 98% 
                 59% 
               
               
                   
                 490 
                 100% 
                 100% 
                 100% 
                 98% 
                 63% 
               
               
                   
                   
               
            
           
         
       
     
     For the data in Table 1, 100% means that each primer in a primer pool forms a dimer with at least one of the other primers in the same primer pool and the length of the dimer is no shorter than the indicated number; 20% means that 20% of the primers in a primer pool forms dimers in the primer pool and the length of the dimer is no shorter than the indicated number. 
     
       
         
           
               
             
               
                 TABLE 2: 
               
             
            
               
                   
               
               
                 Relationship between the number of the primers and 
               
               
                 the dimer length in the 3′ terminal of the primer 
               
            
           
           
               
               
            
               
                 Numbers of 
                 Dimer Length 
               
            
           
           
               
               
               
               
               
               
            
               
                 Primer Pairs 
                 4 bp 
                 5 bp 
                 6 bp 
                 7 bp 
                 8 bp 
               
               
                   
               
               
                  10 
                  50% 
                  10% 
                  0% 
                  0% 
                 0% 
               
               
                  30 
                  90% 
                  50% 
                 13% 
                  7% 
                 7% 
               
               
                  50 
                  98% 
                  56% 
                 26% 
                  2% 
                 0% 
               
               
                  70 
                  97% 
                  67% 
                 23% 
                  7% 
                 0% 
               
               
                  90 
                 100% 
                  80% 
                 33% 
                 12% 
                 6% 
               
               
                 110 
                 100% 
                  88% 
                 50% 
                  7% 
                 3% 
               
               
                 130 
                 100% 
                  82% 
                 40% 
                 10% 
                 4% 
               
               
                 150 
                 100% 
                  91% 
                 41% 
                 13% 
                 2% 
               
               
                 170 
                 100% 
                  95% 
                 49% 
                 15% 
                 4% 
               
               
                 190 
                 100% 
                  94% 
                 54% 
                 15% 
                 3% 
               
               
                 210 
                 100% 
                  94% 
                 50% 
                 14% 
                 3% 
               
               
                 230 
                 100% 
                  97% 
                 59% 
                 20% 
                 6% 
               
               
                 250 
                 100% 
                  99% 
                 58% 
                 21% 
                 7% 
               
               
                 270 
                 100% 
                 100% 
                 69% 
                 23% 
                 6% 
               
               
                 290 
                 100% 
                  99% 
                 65% 
                 19% 
                 5% 
               
               
                 310 
                 100% 
                  99% 
                 67% 
                 22% 
                 6% 
               
               
                 330 
                 100% 
                  99% 
                 70% 
                 24% 
                 4% 
               
               
                 350 
                 100% 
                 100% 
                 71% 
                 25% 
                 7% 
               
               
                 370 
                 100% 
                 100% 
                 73% 
                 26% 
                 6% 
               
               
                 390 
                 100% 
                 100% 
                 73% 
                 27% 
                 7% 
               
               
                 410 
                 100% 
                 100% 
                 75% 
                 31% 
                 10%  
               
               
                 430 
                 100% 
                 100% 
                 77% 
                 36% 
                 8% 
               
               
                 450 
                 100% 
                 100% 
                 79% 
                 34% 
                 7% 
               
               
                 470 
                 100% 
                 100% 
                 81% 
                 31% 
                 9% 
               
               
                 490 
                 100% 
                 100% 
                 82% 
                 36% 
                 9% 
               
               
                   
               
            
           
         
       
     
     For the data in Table 2, 100% means that each primer in a primer pool forms a dimer from its 3′ terminal with at least one of other primers in the same primer pool and the length of the dimer is no shorter than the indicated number, 10% means that 10% of the primers in a primer pool forms dimers in the primer pool and the length of the dimer is no shorter than the indicated number. 
     In some embodiments, there are no more than 20 complementary nucleotide pairings between any two primers. In some embodiments, there are no more than 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 complementary nucleotide pairing between any two primers. In some embodiments, there are no more than 20 consecutive complementary nucleotide pairings between any two primers. In some embodiments, there are no more than 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 consecutive complementary nucleotide pairings between any two primers. In some embodiments, the above mentioned complementary nucleotides pairings are within a region from the 1 st  nucleotide at the 3′ terminal of a primer to the 20 th , 19 th , 18 th , 17 th , 16 th , 15 th , 14 th , 13 th , 12 th , 11 th , 10 th , 9 th  or 8 th  nucleotide from the 3′ terminal of the primer. In some embodiments, there are no more than 7, 6 or 5 consecutive complementary nucleotide pairings within a region from the 1 st  nucleotide at the 3′ terminal of a primer to the 20 th , 19 th , 18 th , 17 th , 16 th , 15 th , 14 th , 13 th , 12 th , 11 th , 10 th , 9 th  or 8 th  nucleotide from the 3′ terminal of the primer. In some embodiments, when calculating the number of parings between two primers, the common tailing sequence is not counted. 
     In some embodiments, there are no more than 20 complementary nucleotide pairings and no more than 50% sequence complementarity between any two primers. In some embodiments, there are no more than 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 complementary nucleotide pairings and no more than 45%, 40%, 35%, 30%, 25% or 20% sequence complementarity between any two primers. In some embodiments, when calculating the percent complementarity between two primers, the common tailing sequence is not counted. 
     The term “nucleotide complementarity” or “complementarity” when in reference to nucleotide as used in the present disclosure refers to a nucleotide on a nucleic acid chain is capable of base pairing with another nucleotide on another nucleic acid chain. For example, in DNA, adenine (A) is complementary to thymine (T), and guanine (G) is complementary to cytosine (C). For another example, in RNA, adenine (A) is complementary to uracil (U), and guanine (G) is complementary to cytosine (C). 
     The term “percent complementarity” as used in the present disclosure refers to the percentage of nucleotide residues in a nucleic acid molecule that have complementarity with nucleotide residues of another nucleic acid molecule when the two nucleic acid molecules are annealed. Percent complementarity is calculated by dividing the number of nucleotides of the first nucleic acid that are complementary to nucleotides at corresponding positions in the second nucleic acid by the total length of the first nucleic acid. 
     Percent complementarity of a nucleic acid or the number of nucleotides of a nucleic acid that is complementary to another nucleic acid can also be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 215, 403-410, 1990; Zhang and Madden, Genome Res., 7, 649-656, 1997). 
     For example, primer 1 in which 18 of 20 nucleotides of the primer 1 have complementarity with 18 nucleotides of primer 2 would have 90% sequence complementarity. In this example, the complementary nucleotides may be contiguous to each other or interspersed with non-complementary nucleotides. 
     The term “x nucleotide pairings” as used in the present disclosure refers to the number of nucleotide residues in a nucleic acid molecule that has complementarity with the corresponding nucleotides of another nucleic acid molecule when the two nucleic acid molecules are annealed. For example, “18 nucleotide pairings” means 18 nucleotide residues of a first nucleic acid molecule has complementarity with 18 nucleotide residues of a second nucleic acid molecule. In this example, the complementary nucleotides may be contiguous to each other or interspersed with non-complementary nucleotides. 
     Nucleic Acid Polymerase 
     In some embodiments, the nucleic acid polymerase may be selected from the family of DNA polymerases like  E. coli  DNA polymerase I (such as  E. coli  DNA polymerase I, Taq DNA polymerase, Tth DNA polymerase, TfI DNA polymerase and others). This polymerase may contain the naturally occurring wild-type sequences or modified variants and fragments thereof. 
     In some embodiments, the nucleic acid polymerase may be selected from modified DNA polymerases of the family of DNA polymerases like  E. coli  DNA polymerase I, e.g., N-terminal deletions of the DNA polymerases, such as Klenow fragment of  E. coli  DNA polymerase I, N-terminal deletions of Taq polymerase (including the Stoffel fragment of Taq DNA polymerase, Klentaq-235, and Klentaq-278) and others. 
     In some embodiments, the nucleic acid polymerase includes, but is not limited to, thermostable DNA polymerases. Examples of thermostable DNA polymerases include, but are not limited to: Tth DNA polymerase, TfI DNA polymerase, Taq DNA polymerase, N-terminal deletions of Taq polymerase (e.g., Stoffel fragment of DNA polymerase, Klentaq-235, and Klentaq-278). Other DNA polymerases include KlenTaqi, Taquenase™ (Amersham), Ad-vanTaq™ (Clontech), GoTaq, GoTaq Flexi (Promega), and KlenTaq-S DNA polymerase. 
     In some embodiments, the nucleic acid polymerase may be commercially available DNA polymerase mixtures, including but are not limited to, TaqLA, TthLA or Expand High Fidelitypius Enzyme Blend (Roche); TthXL Klen TaqLA (Perkin-Elmer); ExTaq® (Takara Shuzo); Elongase® (Life Technologies); Advantage™ KlenTaq, Advantage™ Tth and Advantage2™ (Clontech); TaqExtender™ (Stratagene); Expand™ Long Template and Expand™ High Fidelity (Boehringer Mannheim); and TripleMaster™ Enzyme Mix (Eppendorf). 
     For further decreasing the amplification of undesired nucleic acid, one or more additional polymerase can be added into the reaction mixture. In some embodiments, the reaction mixture comprises high fidelity polymerase. In some embodiments, the high fidelity polymerase is PFU DNA Polymerase, Klentaq-1, Vent, or Deep Vent. 
     De-Blocking Agent 
     De-blocking agent can be selected according to the blocking group contained in the blocking primer. De-blocking agent can be any agent that may result in de-blocking the block group in the blocking primer under the condition of amplifying the target nucleic acid, when the nucleotide with the blocking group in the blocking primer is complementary to the corresponding nucleotide in the target nucleic acid. In some embodiments, the de-blocking agent is pyrophosphate, CS5 DNA polymerase with the mutations selected from G46E, L329A, Q601R, D640G, I669F, S671F, E678G or the combination thereof. In some embodiments, the de-blocking agent is ampliTaq or KlenTaq polymerase with F667Y mutation, or RNase H2. 
     In some embodiments, the de-blocking agent is pyrophosphate, when the blocking group is 2′, 3′-dideoxynucleotide. In some embodiments, the de-blocking agent is CS5 DNA polymerase with the mutations selected from G46E, L329A, Q601R, D640G, I669F, S671F, E678G or the combination thereof (e.g., those DNA polymerases shown in U.S. 20070154914), when the blocking group is 2′-O—PO 3  nucleotide. In some embodiments, the blocking group is 2′-O—PO 3  nucleotide and the de-blocking agent is ampliTaq or KlenTaq polymerase with F667Y mutation, when the blocking group is 2′-O—PO 3  nucleotide. In some embodiments, the de-blocking agent is RNase H2, when the blocking group is ribonucleotide residue. 
     Step of Incubating the Reaction Mixture Under a Condition for Amplification of the Target Nucleic Acid 
     Incubation of the reaction mixture of the present disclosure can be conducted in a multi-cycle process employing several alternating heating and cooling steps to amplify the DNA (see U.S. Pat. Nos. 4,683,202 and 4,683,195). In some embodiments, the incubation comprises the steps of denaturing the target nucleic acid; annealing the primers with the target nucleic acid to allow the formation of a target nucleic acid-primer hybrid; and incubating the target nucleic acid-primer hybrid to allow the nucleic acid polymerase to amplify the target nucleic acid. 
     An example of amplification process is briefly described below. First, a reaction mixture is heated to a temperature sufficient to denature the double stranded target DNA into its two single strands. The temperature of the reaction mixture is then decreased to allow specific single stranded primers to anneal to their respective complementary single-stranded target DNA. Following the annealing step, the temperature is maintained or adjusted to a temperature optimum of the DNA polymerase being used, which allows incorporation of complementary nucleotides at the 3′ ends of the annealed oligonucleotide primers thereby recreating double stranded target DNA. Using a heat-stable DNA polymerase, the cycle of denaturing, annealing and extension may be repeated as many times as necessary to generate a desired product, without the addition of polymerase after each heat denaturation (see “Current Protocols in Molecular Biology”, F. M. Ausubel et al., John Wiley and Sons, Inc., 1998). 
     In some embodiments, denaturing the target nucleic acid is conducted at about 90° C.-100° C. for from about 10 seconds to 10 minutes, preferably for the first circle for from about 1 to 8 minutes. In some embodiments, annealing the primers with the target nucleic acid is conducted at about 5° C.-60° C. for from about 3 seconds to 10 minutes. In some embodiments, incubating the nucleic acid-primer hybrid to allow the nucleic acid polymerase to amplify the target nucleic acid is conducted at about 60° C.-90° C. for from about 1 minute to 15 minutes. 
     In some embodiments, the incubation step is repeated at least 1 time, 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times or 40 times. In some embodiments, the incubation step is repeated from about 20 times to about 50 times. 
     In some embodiments, the nucleic acid other than the target nucleic acid is not amplified in step (b) substantially. In some embodiments, the molar percentage of undesired nucleic acid in the product obtained after the incubation step is less than 20%, 15%, 10%, 5%, 3%, 2% or 1%. 
     The amplification method of the present disclosure can be used to construct DNA sequencing library. In some embodiments, the product obtained from the incubation step can be used as DNA sequencing library directly without enzyme digestion to reduce undesired amplification product. In some embodiments, the product obtained from the incubation step can be used as DNA sequencing library after the ligation of adaptor tags, but without enzyme digestion to reduce undesired amplification product. 
     “DNA sequencing library” as described in the present disclosure refers to a collection of DNA segments, in an abundance that can be sequenced, wherein one end or both ends of each segment in the collection of DNA segments contains a specific sequence partly or completely complementary to the primers used in sequencing, and thereby can be directly used in the subsequent DNA sequencing. 
     Some examples for construction of DNA sequencing library are shown in  FIGS.  1 - 4  and  6 - 7   . 
     In some embodiments, the method is used for selective enrichment of mutant nucleic acid in a sample comprising wildtype and mutant nucleic acid. 
     Some examples for selective enrichment of mutant nucleic acid are shown in  FIG.  5 - 7   . 
     Another aspect of the present disclosure provides method of sequencing a target nucleic acid, wherein the method comprises: (a) providing a reaction mixture comprising: (i) a nucleic acid sample, (ii) at least 20 different pairs of primers, wherein at least one primer of each primer pair is complementary to a portion of the target nucleic acid and comprises a blocking group capable of blocking polymerase extension (“blocking primer”), (iii) nucleic acid polymerase, and (iv) de-blocking agent capable of enabling polymerization of the target nucleic acid by said nucleic acid polymerase; (b) incubating the reaction mixture under a condition for amplification of the target nucleic acid; (c) determining the sequence of the products obtained from step (b). 
     The terms “sequencing” as used in the present disclosure refers to any and all biochemical methods that may be used to determine the identity and order of nucleotide bases including but not limited to adenine, guanine, cytosine and thymine, in one or more molecules of DNA. In some embodiments, the method is use for sequencing by capillary electrophoresis, PCR or high throughput sequencing (e.g., next-generation sequencing (NGS)). 
     Yet another aspect of the present disclosure provides a method of sequencing a target nucleic acid, wherein the method comprises: (a) providing a reaction mixture comprising: (i) a nucleic acid sample, (ii) at least 20 different pairs of primers, wherein at least one primer of each primer pairs is complementary to a portion of the target nucleic acid and comprises a blocking group capable of blocking polymerase extension (“blocking primer”), (iii) nucleic acid polymerase, and (iv) de-blocking agent capable of enabling polymerization of the target nucleic acid by said nucleic acid polymerase; (b) incubating the reaction mixture under a condition for amplification of the target nucleic acid; (c) adding adaptor tag, molecular index tag and/or sample index tag to the product obtained from step (b); (d) determining the sequence of the products obtained from step (c). 
     In some embodiments, in the step (c), adaptor tag, molecular index tag and/or sample index tag is attached to the target nucleic acid obtained from step (b). The adaptor tag, molecular index tag and/or sample index tag can be attached according to the method mentioned above. 
     Yet another aspect of the present disclosure provides a kit of amplifying a target nucleic acid, wherein the kit comprises: (i) at least 20 different pairs of primers, wherein at least one primer of each primer pairs is complementary to a portion of the target nucleic acid and comprises a blocking group capable of blocking polymerase extension, (ii) nucleic acid polymerase, and (iii) de-blocking agent capable of enabling polymerization of the target nucleic acid by said nucleic acid polymerase. 
     In some embodiments, the kit further comprises one or more agents selected from dNTPs, Mg 2+  (e.g., MgCl 2 ), Bovin Serum Albumin, pH buffer (e.g., Tris HCL), glycerol, DNase inhibitor, RNase, SO42 − , Cl − , K + , Ca 2+ , Na + , and (NH 4 ) + . 
     In some embodiments, the kit further comprises an instruction showing how to conduct the amplification of the target nucleic acid (such as showing those methods of the present disclosure). 
     Yet another aspect of the present disclosure provides a method of amplifying a target nucleic acid, wherein the method comprises: (a) providing a reaction mixture comprising: (i) a nucleic acid sample, (ii) at least one primer that is complementary to a portion of the target nucleic acid and comprises a blocking group capable of blocking polymerase extension (“blocking primer”), wherein the blocking primer is modified so as to decrease the amplification of undesired nucleic acid, (iii) nucleic acid polymerase, and (iv) de-blocking agent capable of enabling polymerization of the target nucleic acid by said nucleic acid polymerase; (b) incubating the reaction mixture under a condition for amplification of the target nucleic acid. 
     Yet another aspect of the present disclosure provides a kit of amplifying a target nucleic acid, wherein the kit comprises: (i) at least one primer that is complementary to a portion of the target nucleic acid and comprises a blocking group capable of blocking polymerase extension (“blocking primer”), wherein the blocking primer is modified so as to decrease the amplification of undesired nucleic acid, (ii) nucleic acid polymerase, and (iii) de-blocking agent capable of enabling polymerization of the target nucleic acid by said nucleic acid polymerase. 
     Any embodiment following any aspect of the present disclosure can be applied to other aspects of the present disclosure, as long as the resulted embodiments are possible or reasonable for a person skilled in the art. 
     It is understood that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include the plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to a “bridge probe” is a reference to one or more bridge probes, and includes equivalents thereof known to those skilled in the art and so forth. 
     All publications and patents cited in this specification are herein incorporated by reference to their entirety. 
     EXAMPLES 
     The invention will be more readily understood with reference to the following examples, which are not to be interpreted in any way as limiting the scope of the invention. All specific compositions, materials, and methods described below, in whole or in part, fall within the scope of the invention. These specific compositions, materials, and methods are not intended to limit the invention, but merely to illustrate specific embodiments falling within the scope of the invention. One skilled in the art may develop equivalent compositions, materials, and methods without the exercise of inventive capacity and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures herein described while still remaining within the bounds of the invention. It is the intention of the inventors that such variations are included within the scope of the invention. 
     Example 1. Multiplex PCR Amplification of Genomic DNA Target 
     A multiplex polymerase chain reaction was performed to selectively amplify 196 amplicons (the products amplified from target nucleic acid regions) across human genomic DNA. Each primer pair contains two primers with dideoxynucleoside terminated at its 3′ end and can selectively hybridize target nucleic acid. The sequence of each primer pair is shown in Table 3. The boldfaced sequences in each primer are the sequences for the following step of the library construction and other sequences in each primer are the sequences for selectively hybridizing target nucleic acid. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Amplicons and corresponding primer pairs 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Assay_ 
                 Target_ 
                 Forward_ 
                 SEQ. ID 
                 Forward_ 
                 Reverse_ 
                 SEQ. ID 
                 Reverse_ 
                   
               
               
                 ID 
                 Gene 
                 Primer_ID 
                 NO: 
                 Primer 
                 Primer_ID 
                 NO: 
                 Primer 
                 Chr 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 1 
                 PDGFRA 
                 1F 
                 1 
                 
                   ACACTCTTTCCC 
                 
                 1R 
                 2 
                 
                   GTGACTGGAGT 
                 
                 4 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT AAA 
                   
                   
                   TCTTCCGATCT CA 
                   
               
               
                   
                   
                   
                   
                 ACAAGCTCTCAT 
                   
                   
                 TGTGGTTGTGAA 
                   
               
               
                   
                   
                   
                   
                 GTCTGAACT 
                   
                   
                 AACTGTTCAA 
                   
               
               
                   
               
               
                 2 
                 CDKN2A 
                 2F 
                 3 
                 
                   ACACTCTTTCCC 
                 
                 2R 
                 4 
                 
                   GTGACTGGAGT 
                 
                 9 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CAT 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 GGTTACTGCCTC 
                   
                   
                 ACCAGCGTGTCC 
                   
               
               
                   
                   
                   
                   
                 TGGTG 
                   
                   
                 AGGAA 
                   
               
               
                   
               
               
                 3 
                 SMARCB1 
                 3F 
                 5 
                 
                   ACACTCTTTCCC 
                 
                 3R 
                 6 
                 
                   GTGACTGGAGT 
                 
                 22 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT ACA 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 TGGAGATCGATG 
                   
                   
                 CTGCCTGTCAGG 
                   
               
               
                   
                   
                   
                   
                 GGCA 
                   
                   
                 CAGAT 
                   
               
               
                   
               
               
                 4 
                 TP53 
                 4F 
                 7 
                 
                   ACACTCTTTCCC 
                 
                 4R 
                 8 
                 
                   GTGACTGGAGT 
                 
                 17 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CBG 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 AGGTCACTCACC 
                   
                   
                 GGGAGAAGTAA 
                   
               
               
                   
                   
                   
                   
                 TGG 
                   
                   
                 GTATATACacagt 
                   
               
               
                   
               
               
                 5 
                 RB1 
                 5F 
                 9 
                 
                   ACACTCTTTCCC 
                 
                 5R 
                 10 
                 
                   GTGACTGGAGT 
                 
                 13 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT AGA 
                   
                   
                   TCTTCCGATCT AT 
                   
               
               
                   
                   
                   
                   
                 ACAAAACCATGT 
                   
                   
                 TGTAACAGCATA 
                   
               
               
                   
                   
                   
                   
                 AATAAAATTCTG 
                   
                   
                 CAAGGATCTTCC 
                   
               
               
                   
                   
                   
                   
                 A 
                   
                   
                   
                   
               
               
                   
               
               
                 6 
                 SMAD4 
                 6F 
                 11 
                 
                   ACACTCTTTCCC 
                 
                 6R 
                 12 
                 
                   GTGACTGGAGT 
                 
                 18 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TTCA 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 TTTGTTTTCCCCT 
                   
                   
                 AGTAATGGTAGG 
                   
               
               
                   
                   
                   
                   
                 TTAAACAATTA 
                   
                   
                 TAATCTGTTTCTT 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 AC 
                   
               
               
                   
               
               
                 7 
                 ATM 
                 7F 
                 13 
                 
                   ACACTCTTTCCC 
                 
                 7R 
                 14 
                 
                   GTGACTGGAGT 
                 
                 11 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TGA 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 GGGTACCAGAG 
                   
                   
                 ATTTTTATGTACT 
                   
               
               
                   
                   
                   
                   
                 ACAGT 
                   
                   
                 TTTCATTCCCTGA 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 A 
                   
               
               
                   
               
               
                 8 
                 RB1 
                 8F 
                 15 
                 
                   ACACTCTTTCCC 
                 
                 8R 
                 16 
                 
                   GTGACTGGAGT 
                 
                 13 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT YTG 
                   
                   
                   TCTTCCGATCT CT 
                   
               
               
                   
                   
                   
                   
                 TTATTTAGTTTTG 
                   
                   
                 CCACACACTCCA 
                   
               
               
                   
                   
                   
                   
                 AAACACAGAGA 
                   
                   
                 GTTAGGTA 
                   
               
               
                   
                   
                   
                   
                 A 
                   
                   
                   
                   
               
               
                   
               
               
                 9 
                 ATM 
                 9F 
                 17 
                 
                   ACACTCTTTCCC 
                 
                 9R 
                 18 
                 
                   GTGACTGGAGT 
                 
                 11 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TCTA 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 TGCAAGATACAC 
                   
                   
                 TGCACTGAAAG 
                   
               
               
                   
                   
                   
                   
                 AGTAAAGGTTC 
                   
                   
                 AGGATCGT 
                   
               
               
                   
               
               
                 10 
                 KDR 
                 10F 
                 19 
                 
                   ACACTCTTTCCC 
                 
                 10R 
                 20 
                 
                   GTGACTGGAGT 
                 
                 4 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CTCT 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 GACAAGAGCAT 
                   
                   
                 GTTTCAGATCCA 
                   
               
               
                   
                   
                   
                   
                 GCCATAG 
                   
                   
                 CAGGGATTG 
                   
               
               
                   
               
               
                 11 
                 JAK3 
                 11F 
                 21 
                 
                   ACACTCTTTCCC 
                 
                 11R 
                 22 
                 
                   GTGACTGGAGT 
                 
                 19 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CCA 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 CCTGATTGCATG 
                   
                   
                 GCACTTCTCCAG 
                   
               
               
                   
                   
                   
                   
                 CCA 
                   
                   
                 CCCAA 
                   
               
               
                   
               
               
                 12 
                 PIK3CA 
                 12F 
                 23 
                 
                   ACACTCTTTCCC 
                 
                 12R 
                 24 
                 
                   GTGACTGGAGT 
                 
                 3 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TGA 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 CRAAGAACAGCT 
                   
                   
                 CTGAATTTGGCT 
                   
               
               
                   
                   
                   
                   
                 CAAAGC 
                   
                   
                 GATCTCAGC 
                   
               
               
                   
               
               
                 13 
                 NPM1 
                 13F 
                 25 
                 
                   ACACTCTTTCCC 
                 
                 13R 
                 26 
                 
                   GTGACTGGAGT 
                 
                 5 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT ATG 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 TCTATGAAGTGT 
                   
                   
                 AAATTTTCCGTC 
                   
               
               
                   
                   
                   
                   
                 TGTGGTTCC 
                   
                   
                 TTATTTCATTTCT 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 GT 
                   
               
               
                   
               
               
                 14 
                 RET 
                 14F 
                 27 
                 
                   ACACTCTTTCCC 
                 
                 14R 
                 28 
                 
                   GTGACTGGAGT 
                 
                 10 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GGT 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 CNGATTCCAGTT 
                   
                   
                 CGCAAAGTGATG 
                   
               
               
                   
                   
                   
                   
                 AAATGG 
                   
                   
                 TGTAAGTGTG 
                   
               
               
                   
               
               
                 15 
                 FGFR1 
                 15F 
                 29 
                 
                   ACACTCTTTCCC 
                 
                 15R 
                 30 
                 
                   GTGACTGGAGT 
                 
                 8 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT ACC 
                   
                   
                 TCTTCCGATCTC 
                   
               
               
                   
                   
                   
                   
                 CTGCTTGCAGGA 
                   
                   
                 CAGTGATGGGTT 
                   
               
               
                   
                   
                   
                   
                 TGG 
                   
                   
                 GTAAACCTC 
                   
               
               
                   
               
               
                 16 
                 FLT3 
                 16F 
                 31 
                 
                   ACACTCTTTCCC 
                 
                 16R 
                 32 
                 
                   GTGACTGGAGT 
                 
                 13 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT ATTT 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 TCGTGGAAGTG 
                   
                   
                 CTTCCCAGCTGG 
                   
               
               
                   
                   
                   
                   
                 GGTTACC 
                   
                   
                 GTCAT 
                   
               
               
                   
               
               
                 17 
                 RB1 
                 17F 
                 33 
                 
                   ACACTCTTTCCC 
                 
                 17R 
                 34 
                 
                   GTGACTGGAGT 
                 
                 13 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TGT 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 GGTTTTAATTTC 
                   
                   
                 CTGCAGCAGATA 
                   
               
               
                   
                   
                   
                   
                 ATCATGTTTCATA 
                   
                   
                 TGTAAGCAAAA 
                   
               
               
                   
               
               
                 18 
                 MLH1 
                 18F 
                 35 
                 
                   ACACTCTTTCCC 
                 
                 18R 
                 36 
                 
                   GTGACTGGAGT 
                 
                 3 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT AAG 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 TAGTGATAAGGT 
                   
                   
                 GACAGATATTTC 
                   
               
               
                   
                   
                   
                   
                 CTATGCCCA 
                   
                   
                 TAGTGGCAGGG 
                   
               
               
                   
               
               
                 19 
                 SMAD4 
                 19F 
                 37 
                 
                   ACACTCTTTCCC 
                 
                 19R 
                 38 
                 
                   GTGACTGGAGT 
                 
                 18 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CCT 
                   
                   
                   TCTTCCGATCT TT 
                   
               
               
                   
                   
                   
                   
                 GTTCACAATGAG 
                   
                   
                 TCCTGTATTTAGA 
                   
               
               
                   
                   
                   
                   
                 CTTGCA 
                   
                   
                 TTGATTTAGTGG 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 T 
                   
               
               
                   
               
               
                 20 
                 CDH1 
                 20F 
                 39 
                 
                   ACACTCTTTCCC 
                 
                 20R 
                 40 
                 
                   GTGACTGGAGT 
                 
                 16 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TCG 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 ACACCCGATTCA 
                   
                   
                 GTTTCATAACCC 
                   
               
               
                   
                   
                   
                   
                 AAGTG 
                   
                   
                 ACAGATCCAT 
                   
               
               
                   
               
               
                 21 
                 ATM 
                 21F 
                 41 
                 
                   ACACTCTTTCCC 
                 
                 21R 
                 42 
                 
                   GTGACTGGAGT 
                 
                 11 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TGC 
                   
                   
                   TCTTCCGATCT TT 
                   
               
               
                   
                   
                   
                   
                 YTTCTGGCTGGA 
                   
                   
                 TTTGGTTTTTAA 
                   
               
               
                   
                   
                   
                   
                 TTTAAAT 
                   
                   
                 AATTAATGTTGG 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 CA 
                   
               
               
                   
               
               
                 22 
                 PTEN 
                 22F 
                 43 
                 
                   ACACTCTTTCCC 
                 
                 22R 
                 44 
                 
                   GTGACTGGAGT 
                 
                 10 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GCR 
                   
                   
                   TCTTCCGATC TT 
                   
               
               
                   
                   
                   
                   
                 TGCAGATAATGA 
                   
                   
                 GACTTGTATGTAT 
                   
               
               
                   
                   
                   
                   
                 CAAGGAA 
                   
                   
                 GTGATGTGTG 
                   
               
               
                   
               
               
                 23 
                 AKT1 
                 23F 
                 45 
                 
                   ACACTCTTTCCC 
                 
                 23R 
                 46 
                 
                   GTGACTGGAGT 
                 
                 14 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CGC 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 CACAGAGAAGT 
                   
                   
                 TGAGAGCCACG 
                   
               
               
                   
                   
                   
                   
                 TGTTGAG 
                   
                   
                 CACACT 
                   
               
               
                   
               
               
                 24 
                 FGFR3 
                 24F 
                 47 
                 
                   ACACTCTTTCCC 
                 
                 24R 
                 48 
                 
                   GTGACTGGAGT 
                 
                 4 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT gCC 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 CCTGAGCGTCAT 
                   
                   
                 AGTTCCACTGCA 
                   
               
               
                   
                   
                   
                   
                 CTG 
                   
                   
                 AGGTGT 
                   
               
               
                   
               
               
                 25 
                 RET 
                 25F 
                 49 
                 
                   ACACTCTTTCCC 
                 
                 25R 
                 50 
                 
                   GTGACTGGAGT 
                 
                 10 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CTCT 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 GTGCTGCATTTC 
                   
                   
                 CACCCACATGTC 
                   
               
               
                   
                   
                   
                   
                 AGAGA 
                   
                   
                 ATCAAAT 
                   
               
               
                   
               
               
                 26 
                 ATM 
                 26F 
                 51 
                 
                   ACACTCTTTCCC 
                 
                 26R 
                 52 
                 
                   GTGACTGGAGT 
                 
                 11 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GAT 
                   
                   
                   TCTTCCGATCT TC 
                   
               
               
                   
                   
                   
                   
                 GAGAAAYTCTCA 
                   
                   
                 AGGAAGTCACT 
                   
               
               
                   
                   
                   
                   
                 GGAAACTCTGT 
                   
                   
                 GATGTGAAG 
                   
               
               
                   
               
               
                 27 
                 FLT3 
                 27F 
                 53 
                 
                   ACACTCTTTCCC 
                 
                 27R 
                 54 
                 
                   GTGACTGGAGT 
                 
                 13 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TTCC 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 ATTCTTACCAAA 
                   
                   
                 CCTAAATTGCTT 
                   
               
               
                   
                   
                   
                   
                 CTCTAAATTTTC 
                   
                   
                 CAGAGATGAAA 
                   
               
               
                   
               
               
                 28 
                 KRAS 
                 28F 
                 55 
                 
                   ACACTCTTTCCC 
                 
                 28R 
                 56 
                 
                   GTGACTGGAGT 
                 
                 12 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GCA 
                   
                   
                   TCTTCCGATCT TT 
                   
               
               
                   
                   
                   
                   
                 GAAAACAGATCT 
                   
                   
                 CCTACTAGGACC 
                   
               
               
                   
                   
                   
                   
                 GTATTTATTTCA 
                   
                   
                 ATAGGTACA 
                   
               
               
                   
               
               
                 29 
                 STK11 
                 29F 
                 57 
                 
                   ACACTCTTTCCC 
                 
                 29R 
                 58 
                 
                   GTGACTGGAGT 
                 
                 19 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CTG 
                   
                   
                   TCTTCCGATCT CA 
                   
               
               
                   
                   
                   
                   
                 CCCGCAGGTACT 
                   
                   
                 TTGTGCACAAGG 
                   
               
               
                   
                   
                   
                   
                 TCT 
                   
                   
                 ACATCAAG 
                   
               
               
                   
               
               
                 30 
                 FLT3 
                 30F 
                 59 
                 
                   ACACTCTTTCCC 
                 
                 30R 
                 60 
                 
                   GTGACTGGAGT 
                 
                 13 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TGG 
                   
                   
                   TCTTCCGATCT TA 
                   
               
               
                   
                   
                   
                   
                 GTATCCATCCGA 
                   
                   
                 GAAAAGAACGT 
                   
               
               
                   
                   
                   
                   
                 GAAACA 
                   
                   
                 GTGAAATAAGCT 
                   
               
               
                   
               
               
                 31 
                 ABL1 
                 31F 
                 61 
                 
                   ACACTCTTTCCC 
                 
                 31R 
                 62 
                 
                   GTGACTGGAGT 
                 
                 9 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GCA 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 ACAAGCCCACTG 
                   
                   
                 AAGAAATACAGC 
                   
               
               
                   
                   
                   
                   
                 TCTATG 
                   
                   
                 CTGACGGTG 
                   
               
               
                   
               
               
                 32 
                 VHL 
                 32F 
                 63 
                 
                   ACACTCTTTCCC 
                 
                 32R 
                 64 
                 
                   GTGACTGGAGT 
                 
                 3 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CCC 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 AGGTCATCTTCT 
                   
                   
                 GCATCCACAGCT 
                   
               
               
                   
                   
                   
                   
                 GCAATC 
                   
                   
                 ACCGA 
                   
               
               
                   
               
               
                 33 
                 ATM 
                 33F 
                 65 
                 
                   ACACTCTTTCCC 
                 
                 33R 
                 66 
                 
                   GTGACTGGAGT 
                 
                 11 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT AAA 
                   
                   
                   TCTTCCGATCT T 
                   
               
               
                   
                   
                   
                   
                 GATCACCTTCAG 
                   
                   
                 GTTACCATTTTCT 
                   
               
               
                   
                   
                   
                   
                 AAGTCACAG 
                   
                   
                 CATTCAGTGTCA 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 T 
                   
               
               
                   
               
               
                 34 
                 KDR 
                 34F 
                 67 
                 
                   ACACTCTTTCCC 
                 
                 34R 
                 68 
                 
                   GTGACTGGAGT 
                 
                 4 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT ATTT 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 TATTTCCTCCCTG 
                   
                   
                 TCAAGAGTAAG 
                   
               
               
                   
                   
                   
                   
                 GAAGTCC 
                   
                   
                 GAAAAGATTCA 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 GACT 
                   
               
               
                   
               
               
                 35 
                 FGFR2 
                 35F 
                 69 
                 
                   ACACTCTTTCCC 
                 
                 35R 
                 70 
                 
                   GTGACTGGAGT 
                 
                 10 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TCA 
                   
                   
                   TCTTCCGATCT TC 
                   
               
               
                   
                   
                   
                   
                 CCATCCTGTGTG 
                   
                   
                 TCCATCTCTGAC 
                   
               
               
                   
                   
                   
                   
                 CAGG 
                   
                   
                 ACCAGA 
                   
               
               
                   
               
               
                 36 
                 NRAS 
                 36F 
                 71 
                 
                   ACACTCTTTCCC 
                 
                 36R 
                 72 
                 
                   GTGACTGGAGT 
                 
                 1 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CAT 
                   
                   
                   TCTTCCGATCT TT 
                   
               
               
                   
                   
                   
                   
                 GTATTGGTCTCT 
                   
                   
                 CAATTTTTATTAA 
                   
               
               
                   
                   
                   
                   
                 CATGGCAC 
                   
                   
                 AAACCACAGGG 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 A 
                   
               
               
                   
               
               
                 37 
                 ERBB4 
                 37F 
                 73 
                 
                   ACACTCTTTCCC 
                 
                 37R 
                 74 
                 
                   GTGACTGGAGT 
                 
                 2 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT ACC 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 AGTGACTAGAAA 
                   
                   
                 GAAACAAGACT 
                   
               
               
                   
                   
                   
                   
                 GATCAAATTCC 
                   
                   
                 CAGAGTTAGGG 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 G 
                   
               
               
                   
               
               
                 38 
                 RB1 
                 38F 
                 75 
                 
                   ACACTCTTTCCC 
                 
                 38R 
                 76 
                 
                   GTGACTGGAGT 
                 
                 13 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TGA 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 CATGTAAAGGAT 
                   
                   
                 AAGATCTAGATG 
                   
               
               
                   
                   
                   
                   
                 AATTGTCAGTGA 
                   
                   
                 CAAGATTATTTTT 
                   
               
               
                   
                   
                   
                   
                 C 
                   
                   
                 GG 
                   
               
               
                   
               
               
                 39 
                 SMO 
                 39F 
                 77 
                 
                   ACACTCTTTCCC 
                 
                 39R 
                 78 
                 
                   GTGACTGGAGT 
                 
                 7 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GTG 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 CAGAACATCAAG 
                   
                   
                 AGGACATGCACA 
                   
               
               
                   
                   
                   
                   
                 TTCAACAGT 
                   
                   
                 GCTACATC 
                   
               
               
                   
               
               
                 40 
                 PIK3CA 
                 40F 
                 79 
                 
                   ACACTCTTTCCC 
                 
                 40R 
                 80 
                 
                   GTGACTGGAGT 
                 
                 3 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TAG 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 GTGGAATGAATG 
                   
                   
                 AAAGGGTGCTA 
                   
               
               
                   
                   
                   
                   
                 GCTGAATTA 
                   
                   
                 AAGAGGTAAAG 
                   
               
               
                   
               
               
                 41 
                 KRAS 
                 41F 
                 81 
                 
                   ACACTCTTTCCC 
                 
                 41R 
                 82 
                 
                   GTGACTGGAGT 
                 
                 12 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TTC 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 GTCCACAAAATG 
                   
                   
                 AGTCATTTTCAG 
                   
               
               
                   
                   
                   
                   
                 ATTCTGAATTAG 
                   
                   
                 CAGGCCTTATA 
                   
               
               
                   
               
               
                 42 
                 SMAD4 
                 42F 
                 83 
                 
                   ACACTCTTTCCC 
                 
                 42R 
                 84 
                 
                   GTGACTGGAGT 
                 
                 18 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                 TTCCGATCTTGG 
                   
                   
                 TCTTCCGATCTT 
                   
               
               
                   
                   
                   
                   
                 YGTTCCATTGCTT 
                   
                   
                 GTCCACAGGAC 
                   
               
               
                   
                   
                   
                   
                 ACTTT 
                   
                   
                 AGAAGC 
                   
               
               
                   
               
               
                 43 
                 PIK3CA 
                 43F 
                 85 
                 
                   ACACTCTTTCCC 
                 
                 43R 
                 86 
                 
                   GTGACTGGAGT 
                 
                 3 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TTCC 
                   
                   
                   TCTTCCGATCT T 
                   
               
               
                   
                   
                   
                   
                 ATACTACTCATGA 
                   
                   
                 GAAAGACGATG 
                   
               
               
                   
                   
                   
                   
                 GGTGTTTATTC 
                   
                   
                 GACAAGTAATGG 
                   
               
               
                   
               
               
                 44 
                 RB1 
                 44F 
                 87 
                 
                   ACACTCTTTCCC 
                 
                 44R 
                 88 
                 
                   GTGACTGGAGT 
                 
                 13 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT AGA 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 AGGCAACTTGAC 
                   
                   
                 ATAATTGAAGAA 
                   
               
               
                   
                   
                   
                   
                 AAGAGAAAT 
                   
                   
                 ATTCATTCATGTG 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 CA 
                   
               
               
                   
               
               
                 45 
                 CSF1R 
                 45F 
                 89 
                 
                   ACACTCTTTCCC 
                 
                 45R 
                 90 
                 
                   GTGACTGGAGT 
                 
                 5 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TTCT 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 GCTCAGAGCTCA 
                   
                   
                 CTGAGCAGCTAT 
                   
               
               
                   
                   
                   
                   
                 AGTTC 
                   
                   
                 GTCACAG 
                   
               
               
                   
               
               
                 46 
                 PTEN 
                 46F 
                 91 
                 
                   ACACTCTTTCCC 
                 
                 46R 
                 92 
                 
                   GTGACTGGAGT 
                 
                 10 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CTG 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 GTATGTATTTAAC 
                   
                   
                 TGAAGATATATTC 
                   
               
               
                   
                   
                   
                   
                 CATGCAGATCC 
                   
                   
                 CTCCAATTCAGG 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 AC 
                   
               
               
                   
               
               
                 47 
                 ATM 
                 47F 
                 93 
                 
                   ACACTCTTTCCC 
                 
                 47R 
                 94 
                 
                   GTGACTGGAGT 
                 
                 11 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TGTT 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 GGAAGCTGCTT 
                   
                   
                 TTATTTGAAGAT 
                   
               
               
                   
                   
                   
                   
                 GGG 
                   
                   
                 AAAGAACTTCRG 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 TGG 
                   
               
               
                   
               
               
                 48 
                 KDR 
                 48F 
                 95 
                 
                   ACACTCTTTCCC 
                 
                 48R 
                 96 
                 
                   GTGACTGGAGT 
                 
                 4 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GCT 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 GAGCATTAGCTT 
                   
                   
                 CTCTTTCTTCCTG 
                   
               
               
                   
                   
                   
                   
                 GCAAGA 
                   
                   
                 AATGCTGAAA 
                   
               
               
                   
               
               
                 49 
                 GNAS 
                 49F 
                 97 
                 
                   ACACTCTTTCCC 
                 
                 49R 
                 98 
                 
                   GTGACTGGAGT 
                 
                 20 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TTC 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 AGGACCTGCTTC 
                   
                   
                 CAGTAAGCCAAC 
                   
               
               
                   
                   
                   
                   
                 GCT 
                   
                   
                 TGTTACCTTTT 
                   
               
               
                   
               
               
                 50 
                 PIK3CA 
                 50F 
                 99 
                 
                   ACACTCTTTCCC 
                 
                 50R 
                 100 
                 
                   GTGACTGGAGT 
                 
                 3 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TGC 
                   
                   
                   TCTTCCGATCT TC 
                   
               
               
                   
                   
                   
                   
                 ACAATAAAACAG 
                   
                   
                 TCAAACAGGAG 
                   
               
               
                   
                   
                   
                   
                 TTAGCCAGA 
                   
                   
                 AAGAAGGATGA 
                   
               
               
                   
               
               
                 51 
                 RB1 
                 51F 
                 101 
                 
                   ACACTCTTTCCC 
                 
                 51R 
                 102 
                 
                   GTGACTGGAGT 
                 
                 13 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TCT 
                   
                   
                   TCTTCCGATCT T 
                   
               
               
                   
                   
                   
                   
                 GCATTGGTGCTA 
                   
                   
                 GTAATAATTAAAT 
                   
               
               
                   
                   
                   
                   
                 AAAGTTTCT 
                   
                   
                 TGGCATTCCTTT 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 GG 
                   
               
               
                   
               
               
                 52 
                 MPL 
                 52F 
                 103 
                 
                   ACACTCTTTCCC 
                 
                 52R 
                 104 
                 
                   GTGACTGGAGT 
                 
                 1 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CATC 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 TAGTGCTGGGCC 
                   
                   
                 ACCAGGTGGAG 
                   
               
               
                   
                   
                   
                   
                 TCA 
                   
                   
                 CCGAAG 
                   
               
               
                   
               
               
                 53 
                 STK11 
                 53F 
                 105 
                 
                   ACACTCTTTCCC 
                 
                 53R 
                 106 
                 
                   GTGACTGGAGT 
                 
                 19 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TGC 
                   
                   
                   TCTTCCGATCT GT 
                   
               
               
                   
                   
                   
                   
                 ATAGCCAGGGCA 
                   
                   
                 AGGCACGTGCTA 
                   
               
               
                   
                   
                   
                   
                 TTG 
                   
                   
                 GGGG 
                   
               
               
                   
               
               
                 54 
                 FGFR1 
                 54F 
                 107 
                 
                   ACACTCTTTCCC 
                 
                 54R 
                 108 
                 
                   GTGACTGGAGT 
                 
                 8 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CTG 
                   
                   
                   TCTTCCGATCT CT 
                   
               
               
                   
                   
                   
                   
                 TTTCTTTCTCCTC 
                   
                   
                 AGTGCAGTTCCA 
                   
               
               
                   
                   
                   
                   
                 TGAAGAGG 
                   
                   
                 GATGAACAC 
                   
               
               
                   
               
               
                 55 
                 TP53 
                 55F 
                 109 
                 
                   ACACTCTTTCCC 
                 
                 55R 
                 110 
                 
                   GTGACTGGAGT 
                 
                 17 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GAA 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 AATGTTTCCTGA 
                   
                   
                 TGACCCGGAAG 
                   
               
               
                   
                   
                   
                   
                 CTCAGAGGG 
                   
                   
                 GCAGTC 
                   
               
               
                   
               
               
                 56 
                 RB1 
                 56F 
                 111 
                 
                   ACACTCTTTCCC 
                 
                 56R 
                 112 
                 
                   GTGACTGGAGT 
                 
                 13 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TTTC 
                   
                   
                   TCTTCCGATCT T 
                   
               
               
                   
                   
                   
                   
                 CTTTGTAGTGTC 
                   
                   
                 GTTGAAGAAGTA 
                   
               
               
                   
                   
                   
                   
                 CATAAATTCTTT 
                   
                   
                 TGATGTATTGTTT 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 GC 
                   
               
               
                   
               
               
                 57 
                 CDH1 
                 57F 
                 113 
                 
                   ACACTCTTTCCC 
                 
                 57R 
                 114 
                 
                   GTGACTGGAGT 
                 
                 16 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CGT 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 CGTAATCACCAC 
                   
                   
                 GGAGGCTGTATA 
                   
               
               
                   
                   
                   
                   
                 ACTGAAAG 
                   
                   
                 CACCATATTGA 
                   
               
               
                   
               
               
                 58 
                 FLT3 
                 58F 
                 115 
                 
                   ACACTCTTTCCC 
                 
                 58R 
                 116 
                 
                   GTGACTGGAGT 
                 
                 13 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TCA 
                   
                   
                   TCTTCCGATCT CT 
                   
               
               
                   
                   
                   
                   
                 CATTGCCCCTGA 
                   
                   
                 TCACCACTTTCC 
                   
               
               
                   
                   
                   
                   
                 CAAC 
                   
                   
                 CGTGG 
                   
               
               
                   
               
               
                 59 
                 PDGFRA 
                 59F 
                 117 
                 
                   ACACTCTTTCCC 
                 
                 59R 
                 118 
                 
                   GTGACTGGAGT 
                 
                 4 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCTT CCT 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 GAGTCATTTCTT 
                   
                   
                 CTATGTGTCGAA 
                   
               
               
                   
                   
                   
                   
                 CCTTTTCC 
                   
                   
                 AGGCAGTGTA 
                   
               
               
                   
               
               
                 60 
                 HNF1A 
                 60F 
                 119 
                 
                   ACACTCTTTCCC 
                 
                 60R 
                 120 
                 
                   GTGACTGGAGT 
                 
                 12 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT ATG 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 AGCTACCAACCA 
                   
                   
                 AGATCCTGTTCC 
                   
               
               
                   
                   
                   
                   
                 AGAAGG 
                   
                   
                 AGGCCTAT 
                   
               
               
                   
               
               
                 61 
                 MET 
                 61F 
                 121 
                 
                   ACACTCTTTCCC 
                 
                 61R 
                 122 
                 
                   GTGACTGGAGT 
                 
                 7 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   T TCCGATCTATTT 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 TGGTCTTGCCAG 
                   
                   
                 CTTTGGAAAGTC 
                   
               
               
                   
                   
                   
                   
                 AGACATG 
                   
                   
                 TGCAAACTCAA 
                   
               
               
                   
               
               
                 62 
                 MET 
                 62F 
                 123 
                 
                   ACACTCTTTCCC 
                 
                 62R 
                 124 
                 
                   GTGACTGGAGT 
                 
                 7 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TTCC 
                   
                   
                   TCTTCCGATCT TC 
                   
               
               
                   
                   
                   
                   
                 CTGCAACAGCTG 
                   
                   
                 TCAATGGGCAAT 
                   
               
               
                   
                   
                   
                   
                 AATC 
                   
                   
                 GAAAATGTA 
                   
               
               
                   
               
               
                 63 
                 MET 
                 63F 
                 125 
                 
                   ACACTCTTTCCC 
                 
                 63R 
                 126 
                 
                   GTGACTGGAGT 
                 
                 7 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GCA 
                   
                   
                   TCTTCCGATCT CA 
                   
               
               
                   
                   
                   
                   
                 GTGCTAACCAAG 
                   
                   
                 TGGAGTATACTT 
                   
               
               
                   
                   
                   
                   
                 TTCTTTCT 
                   
                   
                 TTGTGGTTTGC 
                   
               
               
                   
               
               
                 64 
                 AKT1 
                 64F 
                 127 
                 
                   ACACTCTTTCCC 
                 
                 64R 
                 128 
                 
                   GTGACTGGAGT 
                 
                 14 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT ACR 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 ATGACTTCCTTCT 
                   
                   
                 CAGGATCACCTT 
                   
               
               
                   
                   
                   
                   
                 TGAGGA 
                   
                   
                 GCCGAA 
                   
               
               
                   
               
               
                 65 
                 GNA11 
                 65F 
                 129 
                 
                   ACACTCTTTCCC 
                 
                 65R 
                 130 
                 
                   GTGACTGGAGT 
                 
                 19 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CTG 
                   
                   
                   T   CTTCCGATCT CC 
                   
               
               
                   
                   
                   
                   
                 TGTCCTTTCAGG 
                   
                   
                 ACTGCTTTGAGA 
                   
               
               
                   
                   
                   
                   
                 ATGGTG 
                   
                   
                 ACGTGAC 
                   
               
               
                   
               
               
                 66 
                 GNAS 
                 66F 
                 131 
                 
                   ACACTCTTTCCC 
                 
                 66R 
                 132 
                 
                   GTGACTGGAGT 
                 
                 20 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CYC 
                   
                   
                   TCTTCCGATCT CT 
                   
               
               
                   
                   
                   
                   
                 CACCAGCATGTT 
                   
                   
                 TTGCTTCTGTGT 
                   
               
               
                   
                   
                   
                   
                 TGA 
                   
                   
                 TGTTAGGG 
                   
               
               
                   
               
               
                 67 
                 KIT 
                 67F 
                 133 
                 
                   ACACTCTTTCCC 
                 
                 67R 
                 134 
                 
                   GTGACTGGAGT 
                 
                 4 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TTCT 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 AGTGCATTCAAG 
                   
                   
                 CAATTTAAGGGG 
                   
               
               
                   
                   
                   
                   
                 CACAATGG 
                   
                   
                 ATGTTTAGGCT 
                   
               
               
                   
               
               
                 68 
                 PTPN11 
                 68F 
                 135 
                 
                   ACACTCTTTCCC 
                 
                 68R 
                 136 
                 
                   GTGACTGGAGT 
                 
                 12 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CCA 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 ATGGACTATTTTA 
                   
                   
                 GGCAATTAAAAG 
                   
               
               
                   
                   
                   
                   
                 GAAGAAATGGA 
                   
                   
                 AGAAGAATGGA 
                   
               
               
                   
               
               
                 69 
                 ALK 
                 69F 
                 137 
                 
                   ACACTCTTTCCC 
                 
                 69R 
                 138 
                 
                   GTGACTGGAGT 
                 
                 2 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GTC 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 TCTCGGAGGAA 
                   
                   
                 CAGAGAGGGAT 
                   
               
               
                   
                   
                   
                   
                 GGACTT 
                   
                   
                 GTAACCAAAATT 
                   
               
               
                   
               
               
                 70 
                 JAK3 
                 70F 
                 139 
                 
                   ACACTCTTTCCC 
                 
                 70R 
                 140 
                 
                   GTGACTGGAGT 
                 
                 19 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TGA 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 CCTTAGCAGGAT 
                   
                   
                 CTGTCGGTGAGC 
                   
               
               
                   
                   
                   
                   
                 CCAGG 
                   
                   
                 ACTGA 
                   
               
               
                   
               
               
                 71 
                 NRAS 
                 71F 
                 141 
                 
                   ACACTCTTTCCC 
                 
                 71R 
                 142 
                 
                   GTGACTGGAGT 
                 
                 1 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TGA 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 AAGCTGTACCAT 
                   
                   
                 CAGTTCGTGGGC 
                   
               
               
                   
                   
                   
                   
                 ACCTGTCT 
                   
                   
                 TTGTT 
                   
               
               
                   
               
               
                 72 
                 BRAF 
                 72F 
                 143 
                 
                   ACACTCTTTCCC 
                 
                 72R 
                 144 
                 
                   GTGACTGGAGT 
                 
                 7 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GTC 
                   
                   
                   TCTTCCGATCT TC 
                   
               
               
                   
                   
                   
                   
                 ACAATGTCACCA 
                   
                   
                 TACCAAGTGTTT 
                   
               
               
                   
                   
                   
                   
                 CATTACATACT 
                   
                   
                 TCTTGATAAAAA 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 C 
                   
               
               
                   
               
               
                 73 
                 ATM 
                 73F 
                 145 
                 
                   ACACTCTTTCCC 
                 
                 73R 
                 146 
                 
                   GTGACTGGAGT 
                 
                 11 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT ATTT 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 GACCGTGGAGA 
                   
                   
                 AGAGAGCCAAA 
                   
               
               
                   
                   
                   
                   
                 AGTAGAATC 
                   
                   
                 GTACCATAGGTA 
                   
               
               
                   
               
               
                 74 
                 KRAS 
                 74F 
                 147 
                 
                   ACACTCTTTCCC 
                 
                 74R 
                 148 
                 
                   GTGACTGGAGT 
                 
                 12 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TCAT 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 GTACTGGTCCCT 
                   
                   
                 CAAGAGACAGG 
                   
               
               
                   
                   
                   
                   
                 CATTGC 
                   
                   
                 TTTCTCCATCA 
                   
               
               
                   
               
               
                 75 
                 MET 
                 75F 
                 149 
                 
                   ACACTCTTTCCC 
                 
                 75R 
                 150 
                 
                   GTGACTGGAGT 
                 
                 7 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT ATG 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 ATAGCCGTCTTT 
                   
                   
                 AGAAATGGTTTC 
                   
               
               
                   
                   
                   
                   
                 AACAAGCTC 
                   
                   
                 AAATGAATCTGT 
                   
               
               
                   
               
               
                 76 
                 EGFR 
                 76F 
                 151 
                 
                   ACACTCTTTCCC 
                 
                 76R 
                 152 
                 
                   GTGACTGGAGT 
                 
                 7 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CCA 
                   
                   
                   TCTTCCGATCT CA 
                   
               
               
                   
                   
                   
                   
                 GGAACGTACTG 
                   
                   
                 TTTTCCTGACAC 
                   
               
               
                   
                   
                   
                   
                 GTGAAAAC 
                   
                   
                 CAGGGAC 
                   
               
               
                   
               
               
                 77 
                 TP53 
                 77F 
                 153 
                 
                   ACACTCTTTCCC 
                 
                 77R 
                 154 
                 
                   GTGACTGGAGT 
                 
                 17 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TGT 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 CCCAGAATGCAA 
                   
                   
                 GAGCAGCCTCTG 
                   
               
               
                   
                   
                   
                   
                 GAAGC 
                   
                   
                 GCATT 
                   
               
               
                   
               
               
                 78 
                 SMO 
                 78F 
                 155 
                 
                   ACACTCTTTCCC 
                 
                 78R 
                 156 
                 
                   GTGACTGGAGT 
                 
                 7 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TGT 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 TTTGTGGGCTAC 
                   
                   
                 GGCACTTGCTGC 
                   
               
               
                   
                   
                   
                   
                 AAGAACT 
                   
                   
                 CAGTA 
                   
               
               
                   
               
               
                 79 
                 FBXW7 
                 79F 
                 157 
                 
                   ACACTCTTTCCC 
                 
                 79R 
                 158 
                 
                   GTGACTGGAGT 
                 
                 4 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TAA 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 ACTTACTTTGCCT 
                   
                   
                 CACCTATAAGAA 
                   
               
               
                   
                   
                   
                   
                 GTGACTGC 
                   
                   
                 AGATGTGCAGA 
                   
               
               
                   
               
               
                 80 
                 SMO 
                 80F 
                 159 
                 
                   ACACTCTTTCCC 
                 
                 80R 
                 160 
                 
                   GTGACTGGAGT 
                 
                 7 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GTG 
                   
                   
                   TCTTCCGATCT CT 
                   
               
               
                   
                   
                   
                   
                 GAGAAGATCAA 
                   
                   
                 CACCCTCAGCCT 
                   
               
               
                   
                   
                   
                   
                 CCTGTTTGC 
                   
                   
                 TGGG 
                   
               
               
                   
               
               
                 81 
                 MET 
                 81F 
                 161 
                 
                   ACACTCTTTCCC 
                 
                 81R 
                 162 
                 
                   GTGACTGGAGT 
                 
                 7 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GCC 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 TGAATGATGACA 
                   
                   
                 TCAACAAAAACA 
                   
               
               
                   
                   
                   
                   
                 TTCTTTTCG 
                   
                   
                 ATGTGAGATGTC 
                   
               
               
                   
               
               
                 82 
                 ATM 
                 82F 
                 163 
                 
                   ACACTCTTTCCC 
                 
                 82R 
                 164 
                 
                   GTGACTGGAGT 
                 
                 11 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT ACC 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 AGAGTTTCAACA 
                   
                   
                 AGTGGAAGAAG 
                   
               
               
                   
                   
                   
                   
                 AAGTAGCTG 
                   
                   
                 GCACTGTG 
                   
               
               
                   
               
               
                 83 
                 FGFR2 
                 83F 
                 165 
                 
                   ACACTCTTTCCC 
                 
                 83R 
                 166 
                 
                   GTGACTGGAGT 
                 
                 10 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CGG 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 CAYAGGATGACT 
                   
                   
                 GAGTTAGCACAC 
                   
               
               
                   
                   
                   
                   
                 GTTAC 
                   
                   
                 CAGACTG 
                   
               
               
                   
               
               
                 84 
                 PTEN 
                 84F 
                 167 
                 
                   ACACTCTTTCCC 
                 
                 84R 
                 168 
                 
                   GTGACTGGAGT 
                 
                 10 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT ATTT 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 CCATCCTGCAGA 
                   
                   
                 GGATGGATTCGA 
                   
               
               
                   
                   
                   
                   
                 AGAAGC 
                   
                   
                 CTTAGACTTGA 
                   
               
               
                   
               
               
                 85 
                 VHL 
                 85F 
                 169 
                 
                   ACACTCTTTCCC 
                 
                 85R 
                 170 
                 
                   GTGACTGGAGT 
                 
                 3 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GCT 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 CTTTAACAACCT 
                   
                   
                 AATATCACACTG 
                   
               
               
                   
                   
                   
                   
                 TTGCTTGTC 
                   
                   
                 CCAGGTACTG 
                   
               
               
                   
               
               
                 86 
                 KIT 
                 86F 
                 171 
                 
                   ACACTCTTTCCC 
                 
                 86R 
                 172 
                 
                   GTGACTGGAGT 
                 
                 4 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TCTC 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 TTCCATTGTAGA 
                   
                   
                 TTCTCTCTCCAG 
                   
               
               
                   
                   
                   
                   
                 GCAAATCC 
                   
                   
                 AGTGCTCTAAT 
                   
               
               
                   
               
               
                 87 
                 FBXW7 
                 87F 
                 173 
                 
                   ACACTCTTTCCC 
                 
                 87R 
                 174 
                 
                   GTGACTGGAGT 
                 
                 4 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TATT 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 CAAATAACACCC 
                   
                   
                 GTTCACAACTAT 
                   
               
               
                   
                   
                   
                   
                 AATGAAGAATGT 
                   
                   
                 CAATGAGTTCAT 
                   
               
               
                   
               
               
                 88 
                 EGFR 
                 88F 
                 175 
                 
                   ACACTCTTTCCC 
                 
                 88R 
                 176 
                 
                   GTGACTGGAGT 
                 
                 7 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CTCA 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 TCACGCAGCTCA 
                   
                   
                 AGATAAGGAGC 
                   
               
               
                   
                   
                   
                   
                 TGC 
                   
                   
                 CAGGATCCTC 
                   
               
               
                   
               
               
                 89 
                 SMO 
                 89F 
                 177 
                 
                   ACACTCTTTCCC 
                 
                 89R 
                 178 
                 
                   GTGACTGGAGT 
                 
                 7 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CCC 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 AGCATGTCACCA 
                   
                   
                 CTTCTGGGACTG 
                   
               
               
                   
                   
                   
                   
                 AGATG 
                   
                   
                 GAGTACAG 
                   
               
               
                   
               
               
                 90 
                 FBXW7 
                 90F 
                 179 
                 
                   ACACTCTTTCCC 
                 
                 90R 
                 180 
                 
                   GTGACTGGAGT 
                 
                 4 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT AAG 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 TCCCAACCATGA 
                   
                   
                 TGTCCGATCTGT 
                   
               
               
                   
                   
                   
                   
                 CAAGATTTT 
                   
                   
                 AGATCCACTAA 
                   
               
               
                   
               
               
                 91 
                 PTEN 
                 91F 
                 181 
                 
                   ACACTCTTTCCC 
                 
                 91R 
                 182 
                 
                   GTGACTGGAGT 
                 
                 10 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TGC 
                   
                   
                   TCTTCCGATCT TT 
                   
               
               
                   
                   
                   
                   
                 CAGCTAAAGGT 
                   
                   
                 TGTACTTTACTTT 
                   
               
               
                   
                   
                   
                   
                 GAAGAT 
                   
                   
                 CATTGGGAGA 
                   
               
               
                   
               
               
                 92 
                 SMAD4 
                 92F 
                 183 
                 
                   ACACTCTTTCCC 
                 
                 92R 
                 184 
                 
                   GTGACTGGAGT 
                 
                 18 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CTC 
                   
                   
                   TCTTCCGATCT CA 
                   
               
               
                   
                   
                   
                   
                 CATCAAGTATGA 
                   
                   
                 TCCAGCATCCAC 
                   
               
               
                   
                   
                   
                   
                 TGGTGAAGG 
                   
                   
                 CAAGTAAT 
                   
               
               
                   
               
               
                 93 
                 PIK3CA 
                 93F 
                 185 
                 
                   ACACTCTTTCCC 
                 
                 93R 
                 186 
                 
                   GTGACTGGAGT 
                 
                 3 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TCTT 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 CCACACAATTAA 
                   
                   
                 AATTGCACAATC 
                   
               
               
                   
                   
                   
                   
                 ACAGCATG 
                   
                   
                 CATGAACAGC 
                   
               
               
                   
               
               
                 94 
                 KIT 
                 94F 
                 187 
                 
                   ACACTCTTTCCC 
                 
                 94R 
                 188 
                 
                   GTGACTGGAGT 
                 
                 4 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TAG 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 TGTATTCACAGA 
                   
                   
                 AAACGTGAGTAC 
                   
               
               
                   
                   
                   
                   
                 GACTTGGCA 
                   
                   
                 CCATTCTCTG 
                   
               
               
                   
               
               
                 95 
                 KIT 
                 95F 
                 189 
                 
                   ACACTCTTTCCC 
                 
                 95R 
                 190 
                 
                   GTGACTGGAGT 
                 
                 4 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCTC AT 
                   
                   
                   TCTTCCGATCT T 
                   
               
               
                   
                   
                   
                   
                 GTTTCCAATTTTA 
                   
                   
                 GTCCAAGCTGCC 
                   
               
               
                   
                   
                   
                   
                 GCGAGTGC 
                   
                   
                 TTTTATTGTC 
                   
               
               
                   
               
               
                 96 
                 ATM 
                 96F 
                 191 
                 
                   ACACTCTTTCCC 
                 
                 96R 
                 192 
                 
                   GTGACTGGAGT 
                 
                 11 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TGT 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 GTAGGAAAGGT 
                   
                   
                 TGGATTCCTCTA 
                   
               
               
                   
                   
                   
                   
                 ACAATGATTTCC 
                   
                   
                 AGTGAAAATCAT 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 GA 
                   
               
               
                   
               
               
                 97 
                 SMAD4 
                 97F 
                 193 
                 
                   ACACTCTTTCCC 
                 
                 97R 
                 194 
                 
                   GTGACTGGAGT 
                 
                 18 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GAA 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 GGACTGTTGCA 
                   
                   
                 AAGTAGGCAGC 
                   
               
               
                   
                   
                   
                   
                 GATAGCATC 
                   
                   
                 CTTTATAAAAGC 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 A 
                   
               
               
                   
               
               
                 98 
                 ALK 
                 98F 
                 195 
                 
                   ACACTCTTTCCC 
                 
                 98R 
                 196 
                 
                   GTGACTGGAGT 
                 
                 2 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GGG 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 TGAGGCAGTCTT 
                   
                   
                 GGAAGAAAGGA 
                   
               
               
                   
                   
                   
                   
                 TACTCA 
                   
                   
                 AATGCATTTCCT 
                   
               
               
                   
               
               
                 99 
                 EGFR 
                 99F 
                 197 
                 
                   ACACTCTTTCCC 
                 
                 99R 
                 198 
                 
                   GTGACTGGAGT 
                 
                 7 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GCG 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 AAGCCACACTGA 
                   
                   
                 CTGCCTCCTGGA 
                   
               
               
                   
                   
                   
                   
                 CGT 
                   
                   
                 CTATGTC 
                   
               
               
                   
               
               
                 100 
                 BRAF 
                 100F 
                 199 
                 
                   ACACTCTTTCCC 
                 
                 100R 
                 200 
                 
                   GTGACTGGAGT 
                 
                 7 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TTAC 
                   
                   
                   TCTTCCGATCT GT 
                   
               
               
                   
                   
                   
                   
                 CATCCACAAAAT 
                   
                   
                 AAGTAAAGGAA 
                   
               
               
                   
                   
                   
                   
                 GGATCCAG 
                   
                   
                 AACAGTAGATCT 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 CA 
                   
               
               
                   
               
               
                 101 
                 ABL1 
                 101F 
                 201 
                 
                   ACACTCTTTCCC 
                 
                 101R 
                 202 
                 
                   GTGACTGGAGT 
                 
                 9 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CGA 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 AACTGCCTGGTA 
                   
                   
                 GAGCCAAGTTCC 
                   
               
               
                   
                   
                   
                   
                 GGG 
                   
                   
                 CCATC 
                   
               
               
                   
               
               
                 102 
                 ERBB4 
                 102F 
                 203 
                 
                   ACACTCTTTCCC 
                 
                 102R 
                 204 
                 
                   GTGACTGGAGT 
                 
                 2 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT ACTT 
                   
                   
                   TCTTCCGATCT TC 
                   
               
               
                   
                   
                   
                   
                 ACGTGGACATTT 
                   
                   
                 CACTGTCATTGA 
                   
               
               
                   
                   
                   
                   
                 CTTGACAC 
                   
                   
                 AATTCATGCA 
                   
               
               
                   
               
               
                 103 
                 APC 
                 103F 
                 205 
                 
                   ACACTCTTTCCC 
                 
                 103R 
                 206 
                 
                   GTGACTGGAGT 
                 
                 5 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CAG 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 ACTGCAGGGTTC 
                   
                   
                 CCACTCATGTTTA 
                   
               
               
                   
                   
                   
                   
                 TAGTTTATC 
                   
                   
                 GCAGATGTAC 
                   
               
               
                   
               
               
                 104 
                 FGFR2 
                 104F 
                 207 
                 
                   ACACTCTTTCCC 
                 
                 104R 
                 208 
                 
                   GTGACTGGAGT 
                 
                 10 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CAG 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 TCCGGCTTGGAG 
                   
                   
                 GAGTGGGGATG 
                   
               
               
                   
                   
                   
                   
                 GAT 
                   
                   
                 GGAGAA 
                   
               
               
                   
               
               
                 105 
                 PTEN 
                 105F 
                 209 
                 
                   ACACTCTTTCCC 
                 
                 105R 
                 210 
                 
                   GTGACTGGAGT 
                 
                 10 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT ACC 
                   
                   
                   TCTTCCGATCT T 
                   
               
               
                   
                   
                   
                   
                 ACAGCTAGAACT 
                   
                   
                 GTGCATATTTATT 
                   
               
               
                   
                   
                   
                   
                 TATCAAACC 
                   
                   
                 ACATCGGGGC 
                   
               
               
                   
               
               
                 106 
                 RET 
                 106F 
                 211 
                 
                   ACACTCTTTCCC 
                 
                 106R 
                 212 
                 
                   GTGACTGGAGT 
                 
                 10 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GGC 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 TATGGCACCTGC 
                   
                   
                 AGCCCCACAGA 
                   
               
               
                   
                   
                   
                   
                 AAC 
                   
                   
                 GGTCTC 
                   
               
               
                   
               
               
                 107 
                 ERBB4 
                 107F 
                 213 
                 
                   ACACTCTTTCCC 
                 
                 107R 
                 214 
                 
                   GTGACTGGAGT 
                 
                 2 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TTG 
                   
                   
                   TCTTCCGATCT TT 
                   
               
               
                   
                   
                   
                   
                 CAGTCTTACATTT 
                   
                   
                 TTCCTCCAAAGG 
                   
               
               
                   
                   
                   
                   
                 GACCATGA 
                   
                   
                 TCATCAGTTC 
                   
               
               
                   
               
               
                 108 
                 CTNNB1 
                 108F 
                 215 
                 
                   ACACTCTTTCCC 
                 
                 108R 
                 216 
                 
                   GTGACTGGAGT 
                 
                 3 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TAG 
                   
                   
                   TCTTCCGATCT GT 
                   
               
               
                   
                   
                   
                   
                 CTGATTTGATGG 
                   
                   
                 AAAGGCAATCCT 
                   
               
               
                   
                   
                   
                   
                 AGTTGGAC 
                   
                   
                 GAGGAAGAG 
                   
               
               
                   
               
               
                 109 
                 HNF1A 
                 109F 
                 217 
                 
                   ACACTCTTTCCC 
                 
                 109R 
                 218 
                 
                   GTGACTGGAGT 
                 
                 12 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CTG 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 GGCTCCAACCTC 
                   
                   
                 ACAAGCTGGCCA 
                   
               
               
                   
                   
                   
                   
                 GTC 
                   
                   
                 TGGAC 
                   
               
               
                   
               
               
                 110 
                 PDGFRA 
                 110F 
                 219 
                 
                   ACACTCTTTCCC 
                 
                 110R 
                 220 
                 
                   GTGACTGGAGT 
                 
                 4 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TTG 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 GTAATTCACCAG 
                   
                   
                 CCTTATGACTCA 
                   
               
               
                   
                   
                   
                   
                 TTACCTGTC 
                   
                   
                 AGATGGGAGTT 
                   
               
               
                   
               
               
                 111 
                 STK11 
                 111F 
                 221 
                 
                   ACACTCTTTCCC 
                 
                 111R 
                 222 
                 
                   GTGACTGGAGT 
                 
                 19 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GTG 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 GGTATGGACACG 
                   
                   
                 CAAGGTGAAGG 
                   
               
               
                   
                   
                   
                   
                 TTCATC 
                   
                   
                 AGGTGC 
                   
               
               
                   
               
               
                 112 
                 ATM 
                 112F 
                 223 
                 
                   ACACTCTTTCCC 
                 
                 112R 
                 224 
                 
                   GTGACTGGAGT 
                 
                 11 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GCT 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 GTTCCTCAGTTT 
                   
                   
                 AGGTAATTTGCA 
                   
               
               
                   
                   
                   
                   
                 GTCACTAAA 
                   
                   
                 ATTAACTCTTGAT 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 T 
                   
               
               
                   
               
               
                 113 
                 KDR 
                 113F 
                 225 
                 
                   ACACTCTTTCCC 
                 
                 113R 
                 226 
                 
                   GTGACTGGAGT 
                 
                 4 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT AAC 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 AACACTTGAAAA 
                   
                   
                 GTTTGCACTCCA 
                   
               
               
                   
                   
                   
                   
                 TCTGAGCAG 
                   
                   
                 ATCTCTATCAG 
                   
               
               
                   
               
               
                 114 
                 ERBB2 
                 114F 
                 227 
                 
                   ACACTCTTTCCC 
                 
                 114R 
                 228 
                 
                   GTGACTGGAGT 
                 
                 17 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT AATT 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 CCAGTGGCCATC 
                   
                   
                 ACCCTCTCCTGC 
                   
               
               
                   
                   
                   
                   
                 AAAGT 
                   
                   
                 TAGGA 
                   
               
               
                   
               
               
                 115 
                 FBXW7 
                 115F 
                 229 
                 
                   ACACTCTTTCCC 
                 
                 115R 
                 230 
                 
                   GTGACTGGAGT 
                 
                 4 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT ACC 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 YTGCAATGTTTG 
                   
                   
                 TGTGAATGCAAT 
                   
               
               
                   
                   
                   
                   
                 TAAACACTG 
                   
                   
                 TCCCTGTC 
                   
               
               
                   
               
               
                 116 
                 SMAD4 
                 116F 
                 231 
                 
                   ACACTCTTTCCC 
                 
                 116R 
                 232 
                 
                   GTGACTGGAGT 
                 
                 18 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TCT 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 GATGTCTTCCAA 
                   
                   
                 AATTCACTTACA 
                   
               
               
                   
                   
                   
                   
                 ATCTTTTCT 
                   
                   
                 CCGGGCC 
                   
               
               
                   
               
               
                 117 
                 SMAD4 
                 117F 
                 233 
                 
                   ACACTCTTTCCC 
                 
                 117R 
                 234 
                 
                   GTGACTGGAGT 
                 
                 18 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TTT 
                   
                   
                   TCTTCCGATCT GT 
                   
               
               
                   
                   
                   
                   
                 GATTTGCGTCAG 
                   
                   
                 AGGTGGAATAG 
                   
               
               
                   
                   
                   
                   
                 TGTCAT 
                   
                   
                 CTCCAGC 
                   
               
               
                   
               
               
                 118 
                 EGFR 
                 118F 
                 235 
                 
                   ACACTCTTTCCC 
                 
                 118R 
                 236 
                 
                   GTGACTGGAGT 
                 
                 7 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TTA 
                   
                   
                   TCTTCCGATCT T 
                   
               
               
                   
                   
                   
                   
                 ACGTCTTCCTTCT 
                   
                   
                 GAGTTTCTGCTT 
                   
               
               
                   
                   
                   
                   
                 CTCTCTGT 
                   
                   
                 TGCTGTGTG 
                   
               
               
                   
               
               
                 119 
                 JAK3 
                 119F 
                 237 
                 
                   ACACTCTTTCCC 
                 
                 119R 
                 238 
                 
                   GTGACTGGAGT 
                 
                 19 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CTC 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 ACTGTCTCCAGC 
                   
                   
                 AAATTTTGTGCT 
                   
               
               
                   
                   
                   
                   
                 CATG 
                   
                   
                 CACAGACCT 
                   
               
               
                   
               
               
                 120 
                 IDH1 
                 120F 
                 239 
                 
                   ACACTCTTTCCC 
                 
                 120R 
                 240 
                 
                   GTGACTGGAGT 
                 
                 2 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TTG 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 CCAACATGACTT 
                   
                   
                 CAGAATATTTCG 
                   
               
               
                   
                   
                   
                   
                 ACTTGATCC 
                   
                   
                 TATGGTGCCAT 
                   
               
               
                   
               
               
                 121 
                 APC 
                 121F 
                 241 
                 
                   ACACTCTTTCCC 
                 
                 121R 
                 242 
                 
                   GTGACTGGAGT 
                 
                 5 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT ATG 
                   
                   
                   TCTTCCGATCT TT 
                   
               
               
                   
                   
                   
                   
                 CCTCCAGTTCAG 
                   
                   
                 ATTTCTGCCATG 
                   
               
               
                   
                   
                   
                   
                 GAAAAT 
                   
                   
                 CCAACA 
                   
               
               
                   
               
               
                 122 
                 FGFR2 
                 122F 
                 243 
                 
                   ACACTCTTTCCC 
                 
                 122R 
                 244 
                 
                   GTGACTGGAGT 
                 
                 10 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT AAG 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 TCCTCACCTTGA 
                   
                   
                 GGCTGGGCATC 
                   
               
               
                   
                   
                   
                   
                 GAACC 
                   
                   
                 ACTGTA 
                   
               
               
                   
               
               
                 123 
                 PTEN 
                 123F 
                 245 
                 
                   ACACTCTTTCCC 
                 
                 123R 
                 246 
                 
                   GTGACTGGAGT 
                 
                 10 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT AGG 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 ACCAGAGGAAA 
                   
                   
                 AATGATCTTGAC 
                   
               
               
                   
                   
                   
                   
                 CCTCAG 
                   
                   
                 AAAGCAAATAAA 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 GAC 
                   
               
               
                   
               
               
                 124 
                 SMAD4 
                 124F 
                 247 
                 
                   ACACTCTTTCCC 
                 
                 124R 
                 248 
                 
                   GTGACTGGAGT 
                 
                 18 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GTG 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 ATCTATGCCCGTC 
                   
                   
                 AGTTGTATCACC 
                   
               
               
                   
                   
                   
                   
                 TCTGG 
                   
                   
                 TGGAATTGGTA 
                   
               
               
                   
               
               
                 125 
                 APC 
                 125F 
                 249 
                 
                   ACACTCTTTCCC 
                 
                 125R 
                 250 
                 
                   GTGACTGGAGT 
                 
                 5 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT ACT 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 GAGAGCACTGAT 
                   
                   
                 AATGTAAGCCAG 
                   
               
               
                   
                   
                   
                   
                 GATAAACAC 
                   
                   
                 TCTTTGTGTCA 
                   
               
               
                   
               
               
                 126 
                 TP53 
                 126F 
                 251 
                 
                   ACACTCTTTCCC 
                 
                 126R 
                 252 
                 
                   GTGACTGGAGT 
                 
                 17 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TTG 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 TCCTGCTTGCTT 
                   
                   
                 CTACTCAGGATA 
                   
               
               
                   
                   
                   
                   
                 ACCTC 
                   
                   
                 GGAAAAGAGAA 
                   
               
               
                   
               
               
                 127 
                 ERBB4 
                 127F 
                 253 
                 
                   ACACTCTTTCCC 
                 
                 127R 
                 254 
                 
                   GTGACTGGAGT 
                 
                 2 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TGT 
                   
                   
                   TCTTCCGATCT CT 
                   
               
               
                   
                   
                   
                   
                 GGATAACACATA 
                   
                   
                 GGACATTTTTCC 
                   
               
               
                   
                   
                   
                   
                 CCAGGTGA 
                   
                   
                 ACACAGTTTG 
                   
               
               
                   
               
               
                 128 
                 RB1 
                 128F 
                 255 
                 
                   ACACTCTTTCCC 
                 
                 128R 
                 256 
                 
                   GTGACTGGAGT 
                 
                 13 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GTG 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 ATTTCTAAAATA 
                   
                   
                 AAATTTCAgccgg 
                   
               
               
                   
                   
                   
                   
                 GCAGGCTCTTAT 
                   
                   
                 gcgc 
                   
               
               
                   
               
               
                 129 
                 ATM 
                 129F 
                 257 
                 
                   ACACTCTTTCCC 
                 
                 129R 
                 258 
                 
                   GTGACTGGAGT 
                 
                 11 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TGC 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 TTAATTATTCTGA 
                   
                   
                 AGGTCTTCCAGA 
                   
               
               
                   
                   
                   
                   
                 AGGGCCG 
                   
                   
                 TGTGTAATACATT 
                   
               
               
                   
               
               
                 130 
                 HRAS 
                 130F 
                 259 
                 
                   ACACTCTTTCCC 
                 
                 130R 
                 260 
                 
                   GTGACTGGAGT 
                 
                 11 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CGG 
                   
                   
                   TCTTCCGATCT TC 
                   
               
               
                   
                   
                   
                   
                 TGCGCATGTACT 
                   
                   
                 CAACAGGCACG 
                   
               
               
                   
                   
                   
                   
                 GGT 
                   
                   
                 TCTCC 
                   
               
               
                   
               
               
                 131 
                 PTPN11 
                 131F 
                 261 
                 
                   ACACTCTTTCCC 
                 
                 131R 
                 262 
                 
                   GTGACTGGAGT 
                 
                 12 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TCTT 
                   
                   
                   TCTTCCGATCT AT 
                   
               
               
                   
                   
                   
                   
                 CATGATGTTTCCT 
                   
                   
                 TGAAACACTACA 
                   
               
               
                   
                   
                   
                   
                 TCGTAGG 
                   
                   
                 GCGCAGG 
                   
               
               
                   
               
               
                 132 
                 SMO 
                 132F 
                 263 
                 
                   ACACTCTTTCCC 
                 
                 132R 
                 264 
                 
                   GTGACTGGAGT 
                 
                 7 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CTC 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 CAATGAGACTCT 
                   
                   
                 GGGCAAGACCT 
                   
               
               
                   
                   
                   
                   
                 GTCCTGC 
                   
                   
                 CCTACTT 
                   
               
               
                   
               
               
                 133 
                 KIT 
                 133F 
                 265 
                 
                   ACACTCTTTCCC 
                 
                 133R 
                 266 
                 
                   GTGACTGGAGT 
                 
                 4 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TCTC 
                   
                   
                   TCTTCCGATCT T 
                   
               
               
                   
                   
                   
                   
                 AACCATCTGTGA 
                   
                   
                 GGACTTTTGAGA 
                   
               
               
                   
                   
                   
                   
                 GTCCA 
                   
                   
                 TCCTGGATGAA 
                   
               
               
                   
               
               
                 134 
                 ATM 
                 134F 
                 267 
                 
                   ACACTCTTTCCC 
                 
                 134R 
                 268 
                 
                   GTGACTGGAGT 
                 
                 11 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CAG 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 CTGTTACCTGTTT 
                   
                   
                 GATCCAATGCTG 
                   
               
               
                   
                   
                   
                   
                 GAAAAACATTT 
                   
                   
                 GCCTA 
                   
               
               
                   
               
               
                 135 
                 EGFR 
                 135F 
                 269 
                 
                   ACACTCTTTCCC 
                 
                 135R 
                 270 
                 
                   GTGACTGGAGT 
                 
                 7 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CCA 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 CTACATTGACGG 
                   
                   
                 TGGAAAGTGAA 
                   
               
               
                   
                   
                   
                   
                 CCC 
                   
                   
                 GGAGAACAGAA 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 C 
                   
               
               
                   
               
               
                 136 
                 NOTCH1 
                 136F 
                 271 
                 
                   ACACTCTTTCCC 
                 
                 136R 
                 272 
                 
                   GTGACTGGAGT 
                 
                 9 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GAC 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 CAGCGAGGATG 
                   
                   
                 ACTCAGGAAGCT 
                   
               
               
                   
                   
                   
                   
                 GCAG 
                   
                   
                 CCGGC 
                   
               
               
                   
               
               
                 137 
                 FGFR3 
                 137F 
                 273 
                 
                   ACACTCTTTCCC 
                 
                 137R 
                 274 
                 
                   GTGACTGGAGT 
                 
                 4 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CAA 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 TGTGCTGGTGAC 
                   
                   
                 GGTCATGCCAGT 
                   
               
               
                   
                   
                   
                   
                 CGAG 
                   
                   
                 AGGACG 
                   
               
               
                   
               
               
                 138 
                 FGFR3 
                 138F 
                 275 
                 
                   ACACTCTTTCCC 
                 
                 138R 
                 276 
                 
                   GTGACTGGAGT 
                 
                 4 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GGG 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 ACGACTCCGTGT 
                   
                   
                 TGAGGGGTCCCT 
                   
               
               
                   
                   
                   
                   
                 TTG 
                   
                   
                 AGCAG 
                   
               
               
                   
               
               
                 139 
                 KDR 
                 139F 
                 277 
                 
                   ACACTCTTTCCC 
                 
                 139R 
                 278 
                 
                   GTGACTGGAGT 
                 
                 4 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CCC 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 ACTGGATGCTGC 
                   
                   
                 TTGACTGAACTT 
                   
               
               
                   
                   
                   
                   
                 ACA 
                   
                   
                 CCAAAGCAC 
                   
               
               
                   
               
               
                 140 
                 ABL1 
                 140F 
                 279 
                 
                   ACACTCTTTCCC 
                 
                 140R 
                 280 
                 
                   GTGACTGGAGT 
                 
                 9 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GTC 
                   
                   
                   TCTTCCGATCT TC 
                   
               
               
                   
                   
                   
                   
                 TTGTTGGCAGG 
                   
                   
                 ATCCACAGGTAG 
                   
               
               
                   
                   
                   
                   
                 GGTC 
                   
                   
                 GGGC 
                   
               
               
                   
               
               
                 141 
                 APC 
                 141F 
                 281 
                 
                   ACACTCTTTCCC 
                 
                 141R 
                 282 
                 
                   GTGACTGGAGT 
                 
                 5 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CATC 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 AGCTGAAGATG 
                   
                   
                 GCACCCTAGAAC 
                   
               
               
                   
                   
                   
                   
                 AAATAGGATGTA 
                   
                   
                 CAAATCC 
                   
               
               
                   
                   
                   
                   
                 A 
                   
                   
                   
                   
               
               
                   
               
               
                 142 
                 TP53 
                 142F 
                 283 
                 
                   ACACTCTTTCCC 
                 
                 142R 
                 284 
                 
                   GTGACTGGAGT 
                 
                 17 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT SCC 
                   
                   
                   TCTTCCGATCT AT 
                   
               
               
                   
                   
                   
                   
                 AGTTGCAAACCA 
                   
                   
                 CAGTGAGGAATC 
                   
               
               
                   
                   
                   
                   
                 GAC 
                   
                   
                 AGAGGC 
                   
               
               
                   
               
               
                 143 
                 FGFR3 
                 143F 
                 285 
                 
                   ACACTCTTTCCC 
                 
                 143R 
                 286 
                 
                   GTGACTGGAGT 
                 
                 4 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GTG 
                   
                   
                   TCTTCCGATCT CA 
                   
               
               
                   
                   
                   
                   
                 TCTGTCCTGGGA 
                   
                   
                 TCCCTGTGGAGG 
                   
               
               
                   
                   
                   
                   
                 GTCT 
                   
                   
                 AGCT 
                   
               
               
                   
               
               
                 144 
                 KIT 
                 144F 
                 287 
                 
                   ACACTCTTTCCC 
                 
                 144R 
                 288 
                 
                   GTGACTGGAGT 
                 
                 4 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CTG 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 TTGTGCTTCTATT 
                   
                   
                 ATGATCCTTGCC 
                   
               
               
                   
                   
                   
                   
                 ACAGGCTC 
                   
                   
                 AAAGACAACT 
                   
               
               
                   
               
               
                 145 
                 KDR 
                 145F 
                 289 
                 
                   ACACTCTTTCCC 
                 
                 145R 
                 290 
                 
                   GTGACTGGAGT 
                 
                 4 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CTG 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 GCTTTGAATCATT 
                   
                   
                 GGACTCAGAAC 
                   
               
               
                   
                   
                   
                   
                 AGCGTTAC 
                   
                   
                 CACATCATAAAT 
                   
               
               
                   
               
               
                 146 
                 ERBB4 
                 146F 
                 291 
                 
                   ACACTCTTTCCC 
                 
                 146R 
                 292 
                 
                   GTGACTGGAGT 
                 
                 2 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT AAG 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 GTTTACACATTTT 
                   
                   
                 CATTCAGCAAAC 
                   
               
               
                   
                   
                   
                   
                 AATCCCATTTT 
                   
                   
                 AAGCTCAAAAC 
                   
               
               
                   
               
               
                 147 
                 ATM 
                 147F 
                 293 
                 
                   ACACTCTTTCCC 
                 
                 147R 
                 294 
                 
                   GTGACTGGAGT 
                 
                 11 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT ATTA 
                   
                   
                   TCTTCCGATCT TA 
                   
               
               
                   
                   
                   
                   
                 GGTGGACCACA 
                   
                   
                 AGGTGAGCCTTC 
                   
               
               
                   
                   
                   
                   
                 CAGGA 
                   
                   
                 CCTTC 
                   
               
               
                   
               
               
                 148 
                 RET 
                 148F 
                 295 
                 
                   ACACTCTTTCCC 
                 
                 148R 
                 296 
                 
                   GTGACTGGAGT 
                 
                 10 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT ACT 
                   
                   
                   TCTTCCGATCT TA 
                   
               
               
                   
                   
                   
                   
                 CGTGCTATTTTTC 
                   
                   
                 CGTGAAGAGGA 
                   
               
               
                   
                   
                   
                   
                 CTCACAG 
                   
                   
                 GCCAG 
                   
               
               
                   
               
               
                 149 
                 IDH2 
                 149F 
                 297 
                 
                   ACACTCTTTCCC 
                 
                 149R 
                 298 
                 
                   GTGACTGGAGT 
                 
                 15 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GCC 
                   
                   
                   TCTTCCGATCT CA 
                   
               
               
                   
                   
                   
                   
                 TACCTGGTCGCC 
                   
                   
                 TTGGGACTTTTC 
                   
               
               
                   
                   
                   
                   
                 ATG 
                   
                   
                 CACATCTTCT 
                   
               
               
                   
               
               
                 150 
                 MET 
                 150F 
                 299 
                 
                   ACACTCTTTCCC 
                 
                 150R 
                 300 
                 
                   GTGACTGGAGT 
                 
                 7 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GAC 
                   
                   
                   TCTTCCGATCT CT 
                   
               
               
                   
                   
                   
                   
                 ATGTCTTTCCCC 
                   
                   
                 TTCATCTGTAAA 
                   
               
               
                   
                   
                   
                   
                 ACAATCATA 
                   
                   
                 GGACCGGTTC 
                   
               
               
                   
               
               
                 151 
                 SMARCB1 
                 151F 
                 301 
                 
                   ACACTCTTTCCC 
                 
                 151R 
                 302 
                 
                   GTGACTGGAGT 
                 
                 22 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TACT 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 CATAGGTGGGA 
                   
                   
                 CTAACACTAAGG 
                   
               
               
                   
                   
                   
                   
                 AACTACCTC 
                   
                   
                 GTGCGT 
                   
               
               
                   
               
               
                 152 
                 SRC 
                 152F 
                 303 
                 
                   ACACTCTTTCCC 
                 
                 152R 
                 304 
                 
                   GTGACTGGAGT 
                 
                 20 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CTTC 
                   
                   
                   TCTTCCGATCT CT 
                   
               
               
                   
                   
                   
                   
                 CTGGAGGACTAC 
                   
                   
                 CTGCCTGCCTGC 
                   
               
               
                   
                   
                   
                   
                 TTCACG 
                   
                   
                 TGTT 
                   
               
               
                   
               
               
                 153 
                 ATM 
                 153F 
                 305 
                 
                   ACACTCTTTCCC 
                 
                 153R 
                 306 
                 
                   GTGACTGGAGT 
                 
                 11 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT ATCT 
                   
                   
                   TCTTCCGATCT TC 
                   
               
               
                   
                   
                   
                   
                 AGGATCCAAATT 
                   
                   
                 ATCTTGTACTGG 
                   
               
               
                   
                   
                   
                   
                 TTAGAAGTCAAG 
                   
                   
                 AGAAAATTCTTG 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 TG 
                   
               
               
                   
               
               
                 154 
                 NOTCH1 
                 154F 
                 307 
                 
                   ACACTCTTTCCC 
                 
                 154R 
                 308 
                 
                   GTGACTGGAGT 
                 
                 9 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CAC 
                   
                   
                   TCTTCCGATCT T 
                   
               
               
                   
                   
                   
                   
                 TGCCGGTTGTCA 
                   
                   
                 GACGCCACAGTC 
                   
               
               
                   
                   
                   
                   
                 ATCTC 
                   
                   
                 AGGAC 
                   
               
               
                   
               
               
                 155 
                 KIT 
                 155F 
                 309 
                 
                   ACACTCTTTCCC 
                 
                 155R 
                 310 
                 
                   GTGACTGGAGT 
                 
                 4 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TCA 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 CCTTCTTTCTAAC 
                   
                   
                 AACGTGATTCAT 
                   
               
               
                   
                   
                   
                   
                 CTTTTCTTATGT 
                   
                   
                 TTATTTGTTCAAA 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 GC 
                   
               
               
                   
               
               
                 156 
                 KDR 
                 156F 
                 311 
                 
                   ACACTCTTTCCC 
                 
                 156R 
                 312 
                 
                   GTGACTGGAGT 
                 
                 4 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GAT 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 GCTCACTGTGTG 
                   
                   
                 ATAATTGGGGTC 
                   
               
               
                   
                   
                   
                   
                 TTGCT 
                   
                   
                 CCTCCCT 
                   
               
               
                   
               
               
                 157 
                 RET 
                 157F 
                 313 
                 
                   ACACTCTTTCCC 
                 
                 157R 
                 314 
                 
                   GTGACTGGAGT 
                 
                 10 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CGC 
                   
                   
                   TCTTCCGATCT T 
                   
               
               
                   
                   
                   
                   
                 AGCCTGTACCCA 
                   
                   
                 GCTACCACAAGT 
                   
               
               
                   
                   
                   
                   
                 GTG 
                   
                   
                 TTGCCC 
                   
               
               
                   
               
               
                 158 
                 PTEN 
                 158F 
                 315 
                 
                   ACACTCTTTCCC 
                 
                 158R 
                 316 
                 
                   GTGACTGGAGT 
                 
                 10 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GCT 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 ACGACCCAGTTA 
                   
                   
                 GCTACCTGTTAA 
                   
               
               
                   
                   
                   
                   
                 CCATAGC 
                   
                   
                 AGAATCATCTGG 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 A 
                   
               
               
                   
               
               
                 159 
                 GNAQ 
                 159F 
                 317 
                 
                   ACACTCTTTCCC 
                 
                 159R 
                 318 
                 
                   GTGACTGGAGT 
                 
                 9 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GAG 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 GTGACATTTTCA 
                   
                   
                 AATATAGCACTA 
                   
               
               
                   
                   
                   
                   
                 AAGCAGTG 
                   
                   
                 CTTACAAACTTA 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 GGG 
                   
               
               
                   
               
               
                 160 
                 ERBB4 
                 160F 
                 319 
                 
                   ACACTCTTTCCC 
                 
                 160R 
                 320 
                 
                   GTGACTGGAGT 
                 
                 2 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT AAA 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 GTGGCTAAAGTT 
                   
                   
                 TCCTGAGCAGC 
                   
               
               
                   
                   
                   
                   
                 GATCTGATTGT 
                   
                   
                 MTCCAG 
                   
               
               
                   
               
               
                 161 
                 JAK2 
                 161F 
                 321 
                 
                   ACACTCTTTCCC 
                 
                 161R 
                 322 
                 
                   GTGACTGGAGT 
                 
                 9 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CCTT 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 AGTCTTTCTTTG 
                   
                   
                 CTTTCTCAGAGC 
                   
               
               
                   
                   
                   
                   
                 AAGCAGCA 
                   
                   
                 ATCTGTTTTTG 
                   
               
               
                   
               
               
                 162 
                 ERBB4 
                 162F 
                 323 
                 
                   ACACTCTTTCCC 
                 
                 162R 
                 324 
                 
                   GTGACTGGAGT 
                 
                 2 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT ATA 
                   
                   
                   TCTTCCGATCT T 
                   
               
               
                   
                   
                   
                   
                 ACTCATTCATCG 
                   
                   
                 GAATGGTGTCTG 
                   
               
               
                   
                   
                   
                   
                 CCACATAGG 
                   
                   
                 CATAACAAAGG 
                   
               
               
                   
               
               
                 163 
                 EGFR 
                 163F 
                 325 
                 
                   ACACTCTTTCCC 
                 
                 163R 
                 326 
                 
                   GTGACTGGAGT 
                 
                 7 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GTC 
                   
                   
                   TCTTCCGATCT T 
                   
               
               
                   
                   
                   
                   
                 TCTGTGTTCTTGT 
                   
                   
                 GTATAAGGTAAG 
                   
               
               
                   
                   
                   
                   
                 CCCC 
                   
                   
                 GTCCCTGG 
                   
               
               
                   
               
               
                 164 
                 CSF1R 
                 164F 
                 327 
                 
                   ACACTCTTTCCC 
                 
                 164R 
                 328 
                 
                   GTGACTGGAGT 
                 
                 5 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CATC 
                   
                   
                   TCTTCCGATCT TC 
                   
               
               
                   
                   
                   
                   
                 CATGGAGGAGTT 
                   
                   
                 AGGTGCTCACTA 
                   
               
               
                   
                   
                   
                   
                 GAAGTTT 
                   
                   
                 GAGCTC 
                   
               
               
                   
               
               
                 165 
                 VHL 
                 165F 
                 329 
                 
                   ACACTCTTTCCC 
                 
                 165R 
                 330 
                 
                   GTGACTGGAGT 
                 
                 3 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CTCT 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 TGTTCGTTCCTT 
                   
                   
                 GGAGACTGGAC 
                   
               
               
                   
                   
                   
                   
                 GTACTGAG 
                   
                   
                 ATCGTCAG 
                   
               
               
                   
               
               
                 166 
                 PDGFRA 
                 166F 
                 331 
                 
                   ACACTCTTTCCC 
                 
                 166R 
                 332 
                 
                   GTGACTGGAGT 
                 
                 4 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT AGG 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 CYCCATTTACATC 
                   
                   
                 ACCCAGAGAAG 
                   
               
               
                   
                   
                   
                   
                 ATCA 
                   
                   
                 CCAAAGAAAG 
                   
               
               
                   
               
               
                 167 
                 ATM 
                 167F 
                 333 
                 
                   ACACTCTTTCCC 
                 
                 167R 
                 334 
                 
                   GTGACTGGAGT 
                 
                 11 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT ATA 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 GGAAGTAGAGG 
                   
                   
                 CAGGTACAGTAA 
                   
               
               
                   
                   
                   
                   
                 AAAGTATTCTTC 
                   
                   
                 GTAGGTCATGT 
                   
               
               
                   
                   
                   
                   
                 AG 
                   
                   
                   
                   
               
               
                   
               
               
                 168 
                 SMARCB1 
                 168F 
                 335 
                 
                   ACACTCTTTCCC 
                 
                 168R 
                 336 
                 
                   GTGACTGGAGT 
                 
                 22 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CAC 
                   
                   
                   TCTTCCGATCT CT 
                   
               
               
                   
                   
                   
                   
                 CCCTACACTTGG 
                   
                   
                 GGTAACCAGCCC 
                   
               
               
                   
                   
                   
                   
                 CTG 
                   
                   
                 ATCAG 
                   
               
               
                   
               
               
                 169 
                 STK11 
                 169F 
                 337 
                 
                   ACACTCTTTCCC 
                 
                 169R 
                 338 
                 
                   GTGACTGGAGT 
                 
                 19 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CTC 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 CCCTCGAAATGA 
                   
                   
                 GGAGCCTCATCC 
                   
               
               
                   
                   
                   
                   
                 AGCTA 
                   
                   
                 CTCTG 
                   
               
               
                   
               
               
                 170 
                 HRAS 
                 170F 
                 339 
                 
                   ACACTCTTTCCC 
                 
                 170R 
                 340 
                 
                   GTGACTGGAGT 
                 
                 11 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CCA 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 GGCTCACCTCTA 
                   
                   
                 ACCACCAGCTTA 
                   
               
               
                   
                   
                   
                   
                 TAGTGG 
                   
                   
                 TATTCCGT 
                   
               
               
                   
               
               
                 171 
                 ERBB2 
                 171F 
                 341 
                 
                   ACACTCTTTCCC 
                 
                 171R 
                 342 
                 
                   GTGACTGGAGT 
                 
                 17 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CCT 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 CTCAGCGTACCC 
                   
                   
                 GTGCAGCTGGT 
                   
               
               
                   
                   
                   
                   
                 TTGT 
                   
                   
                 GACACA 
                   
               
               
                   
               
               
                 172 
                 EZH2 
                 172F 
                 343 
                 
                   ACACTCTTTCCC 
                 
                 172R 
                 344 
                 
                   GTGACTGGAGT 
                 
                 7 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT ATAC 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 AATGCCACCTGA 
                   
                   
                 TGCCAGCAATAG 
                   
               
               
                   
                   
                   
                   
                 ATACAGG 
                   
                   
                 ATGCTAGA 
                   
               
               
                   
               
               
                 173 
                 FBXW7 
                 173F 
                 345 
                 
                   ACACTCTTTCCC 
                 
                 173R 
                 346 
                 
                   GTGACTGGAGT 
                 
                 4 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CTC 
                   
                   
                   TCTTCCGATCT T 
                   
               
               
                   
                   
                   
                   
                 CTGCCATCATATT 
                   
                   
                 GCAGAGGGAGA 
                   
               
               
                   
                   
                   
                   
                 GAACACAG 
                   
                   
                 AACAGAAAAAC 
                   
               
               
                   
               
               
                 174 
                 KIT 
                 174F 
                 347 
                 
                   ACACTCTTTCCC 
                 
                 174R 
                 348 
                 
                   GTGACTGGAGT 
                 
                 4 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GTA 
                   
                   
                   TCTTCCGATCT AT 
                   
               
               
                   
                   
                   
                   
                 GAGCATGACCCA 
                   
                   
                 GGACATGAAAC 
                   
               
               
                   
                   
                   
                   
                 TGAGTG 
                   
                   
                 CTGGAGTT 
                   
               
               
                   
               
               
                 175 
                 RB1 
                 175F 
                 349 
                 
                   ACACTCTTTCCC 
                 
                 175R 
                 350 
                 
                   GTGACTGGAGT 
                 
                 13 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GTT 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 CTTCCTCAGACA 
                   
                   
                 CAGGGTAGGTC 
                   
               
               
                   
                   
                   
                   
                 TTCAAACGT 
                   
                   
                 AAAAGTATCCTT 
                   
               
               
                   
               
               
                 176 
                 EGFR 
                 176F 
                 351 
                 
                   ACACTCTTTCCC 
                 
                 176R 
                 352 
                 
                   GTGACTGGAGT 
                 
                 7 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TACC 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 AGATGGATGTGA 
                   
                   
                 GAGTATCCCATC 
                   
               
               
                   
                   
                   
                   
                 ACCCC 
                   
                   
                 TTGGAGAGTC 
                   
               
               
                   
               
               
                 177 
                 ERBB4 
                 177F 
                 353 
                 
                   ACACTCTTTCCC 
                 
                 177R 
                 354 
                 
                   GTGACTGGAGT 
                 
                 2 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TTG 
                   
                   
                   TCTTCCGATCT TT 
                   
               
               
                   
                   
                   
                   
                 CCATTTTGGATAT 
                   
                   
                 GTCCCACGAATA 
                   
               
               
                   
                   
                   
                   
                 ATTCCTTACCT 
                   
                   
                 ATGCGTAAAT 
                   
               
               
                   
               
               
                 178 
                 CDH1 
                 178F 
                 355 
                 
                   ACACTCTTTCCC 
                 
                 178R 
                 356 
                 
                   GTGACTGGAGT 
                 
                 16 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT ACT 
                   
                   
                   TCTTCCGATCT TC 
                   
               
               
                   
                   
                   
                   
                 TGGTTGTGTCGA 
                   
                   
                 TTCAATCCCACC 
                   
               
               
                   
                   
                   
                   
                 TCTCTCT 
                   
                   
                 ACGGTAAT 
                   
               
               
                   
               
               
                 179 
                 STK11 
                 179F 
                 357 
                 
                   ACACTCTTTCCC 
                 
                 179R 
                 358 
                 
                   GTGACTGGAGT 
                 
                 19 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT AGA 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 CACCAAGGACC 
                   
                   
                 CATCGAGGATGA 
                   
               
               
                   
                   
                   
                   
                 GGTG 
                   
                   
                 CATCATCTACA 
                   
               
               
                   
               
               
                 180 
                 ABL1 
                 180F 
                 359 
                 
                   ACACTCTTTCCC 
                 
                 180R 
                 360 
                 
                   GTGACTGGAGT 
                 
                 9 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TCA 
                   
                   
                   TCTTCCGATCT T 
                   
               
               
                   
                   
                   
                   
                 AGTACTTACCCA 
                   
                   
                 GCAGCTCCTTGG 
                   
               
               
                   
                   
                   
                   
                 CTGAAAAGC 
                   
                   
                 TGAGTAA 
                   
               
               
                   
               
               
                 181 
                 PTEN 
                 181F 
                 361 
                 
                   ACACTCTTTCCC 
                 
                 181R 
                 362 
                 
                   GTGACTGGAGT 
                 
                 10 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT AGC 
                   
                   
                   TCTTCCGATCT T 
                   
               
               
                   
                   
                   
                   
                 TCATTTTTGTTAA 
                   
                   
                 GCTTGCAAATAT 
                   
               
               
                   
                   
                   
                   
                 TGGTGGCT 
                   
                   
                 CTTCTAAAACAA 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 CTA 
                   
               
               
                   
               
               
                 182 
                 ATM 
                 182F 
                 363 
                 
                   ACACTCTTTCCC 
                 
                 182R 
                 364 
                 
                   GTGACTGGAGT 
                 
                 11 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TGAT 
                   
                   
                   TCTTCCGATCT CA 
                   
               
               
                   
                   
                   
                   
                 AAATKAGCAGTC 
                   
                   
                 TGGAATGTTGTT 
                   
               
               
                   
                   
                   
                   
                 AGCAGAA 
                   
                   
                 TGCCTACC 
                   
               
               
                   
               
               
                 183 
                 RB1 
                 183F 
                 365 
                 
                   ACACTCTTTCCC 
                 
                 183R 
                 366 
                 
                   GTGACTGGAGT 
                 
                 13 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TTTC 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 TTATTCCCACAGT 
                   
                   
                 CTGCAGAATGAG 
                   
               
               
                   
                   
                   
                   
                 GTATCGG 
                   
                   
                 TATGAACTCAT 
                   
               
               
                   
               
               
                 184 
                 KDR 
                 184F 
                 367 
                 
                   ACACTCTTTCCC 
                 
                 184R 
                 368 
                 
                   GTGACTGGAGT 
                 
                 4 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT AGC 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 TTTAAAAGTTCT 
                   
                   
                 ACCATTCCACTG 
                   
               
               
                   
                   
                   
                   
                 GCTTCCTCA 
                   
                   
                 CAGAAGAAAT 
                   
               
               
                   
               
               
                 185 
                 EGFR 
                 185F 
                 369 
                 
                   ACACTCTTTCCC 
                 
                 185R 
                 370 
                 
                   GTGACTGGAGT 
                 
                 7 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TTCC 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 TCAAAAGAGAA 
                   
                   
                 AATATGTACTACG 
                   
               
               
                   
                   
                   
                   
                 ATCACGCAT 
                   
                   
                 AAAATTCCTATG 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 CC 
                   
               
               
                   
               
               
                 186 
                 NOTCH1 
                 186F 
                 371 
                 
                   ACACTCTTTCCC 
                 
                 186R 
                 372 
                 
                   GTGACTGGAGT 
                 
                 9 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT AAG 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 ATCATCTGCTGG 
                   
                   
                 CAGCCTCTCGGG 
                   
               
               
                   
                   
                   
                   
                 CCGT 
                   
                   
                 TACAT 
                   
               
               
                   
               
               
                 187 
                 TP53 
                 187F 
                 373 
                 
                   ACACTCTTTCCC 
                 
                 187R 
                 374 
                 
                   GTGACTGGAGT 
                 
                 17 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT AGA 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 GGCAAGCAGAG 
                   
                   
                 CCTAGGAGATAA 
                   
               
               
                   
                   
                   
                   
                 GCTG 
                   
                   
                 CACAGGCC 
                   
               
               
                   
               
               
                 188 
                 APC 
                 188F 
                 375 
                 
                   ACACTCTTTCCC 
                 
                 188R 
                 376 
                 
                   GTGACTGGAGT 
                 
                 5 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT AGA 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 GAACGCGGAAT 
                   
                   
                 GCCATTCATACCT 
                   
               
               
                   
                   
                   
                   
                 TGGTCT 
                   
                   
                 CTCAGGAA 
                   
               
               
                   
               
               
                 189 
                 SMAD4 
                 189F 
                 377 
                 
                   ACACTCTTTCCC 
                 
                 189R 
                 378 
                 
                   GTGACTGGAGT 
                 
                 18 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CTTT 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 TTYCTTCCTAAG 
                   
                   
                 GTGCACCTGGA 
                   
               
               
                   
                   
                   
                   
                 GTTGCACA 
                   
                   
                 GATGCT 
                   
               
               
                   
               
               
                 190 
                 SMARCB1 
                 190F 
                 379 
                 
                   ACACTCTTTCCC 
                 
                 190R 
                 380 
                 
                   GTGACTGGAGT 
                 
                 22 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TCTT 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 GTATCTCCTCAG 
                   
                   
                 GACAAGAAGAG 
                   
               
               
                   
                   
                   
                   
                 GGAACAG 
                   
                   
                 AACCTTCCCC 
                   
               
               
                   
               
               
                 191 
                 ERBB2 
                 191F 
                 381 
                 
                   ACACTCTTTCCC 
                 
                 191R 
                 382 
                 
                   GTGACTGGAGT 
                 
                 17 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TACA 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 TGGGTGCTTCCC 
                   
                   
                 GGGCAAGGTTA 
                   
               
               
                   
                   
                   
                   
                 ATTC 
                   
                   
                 GGTGAAG 
                   
               
               
                   
               
               
                 192 
                 NRAS 
                 192F 
                 383 
                 
                   ACACTCTTTCCC 
                 
                 192R 
                 384 
                 
                   GTGACTGGAGT 
                 
                 1 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TAC 
                   
                   
                   TCTTCCGATCT G 
                   
               
               
                   
                   
                   
                   
                 AAAGTGGTTCTG 
                   
                   
                 CGAGCCACATCT 
                   
               
               
                   
                   
                   
                   
                 GATTAGCTG 
                   
                   
                 ACAGTACTTTA 
                   
               
               
                   
               
               
                 193 
                 FGFR3 
                 193F 
                 385 
                 
                   ACACTCTTTCCC 
                 
                 193R 
                 386 
                 
                   GTGACTGGAGT 
                 
                 4 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT CAT 
                   
                   
                   TCTTCCGATCT C 
                   
               
               
                   
                   
                   
                   
                 GTCTTTGCAGCC 
                   
                   
                 CAAGAAAGGCC 
                   
               
               
                   
                   
                   
                   
                 GAGG 
                   
                   
                 TGGGCT 
                   
               
               
                   
               
               
                 194 
                 KIT 
                 194F 
                 387 
                 
                   ACACTCTTTCCC 
                 
                 194R 
                 388 
                 
                   GTGACTGGAGT 
                 
                 4 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT GGT 
                   
                   
                   TCTTCCGATCT A 
                   
               
               
                   
                   
                   
                   
                 GATCTATTTTTCC 
                   
                   
                 GAAACAGGCTG 
                   
               
               
                   
                   
                   
                   
                 CTTTCTCCC 
                   
                   
                 AGTTTTGGTC 
                   
               
               
                   
               
               
                 195 
                 KDR 
                 195F 
                 389 
                 
                   ACACTCTTTCCC 
                 
                 195R 
                 390 
                 
                   GTGACTGGAGT 
                 
                 4 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TAG 
                   
                   
                   TCTTCCGATCT TC 
                   
               
               
                   
                   
                   
                   
                 ACAAGGTCTTCC 
                   
                   
                 CTCCTCCATACA 
                   
               
               
                   
                   
                   
                   
                 TTCCACTT 
                   
                   
                 GGAAACAG 
                   
               
               
                   
               
               
                 196 
                 PIK3CA 
                 196F 
                 391 
                 
                   ACACTCTTTCCC 
                 
                 196R 
                 392 
                 
                   GTGACTGGAGT 
                 
                 3 
               
               
                   
                   
                   
                   
                 
                   TACACGACGCTC 
                 
                   
                   
                 
                   TCAGACGTGTGC 
                 
                   
               
               
                   
                   
                   
                   
                   TTCCGATCT TTCT 
                   
                   
                   TCTTCCGATCT T 
                   
               
               
                   
                   
                   
                   
                 CAATGATGCTTG 
                   
                   
                 GGCTGGACAAC 
                   
               
               
                   
                   
                   
                   
                 GCTC 
                   
                   
                 AAAAATGGA 
               
               
                   
               
            
           
         
       
     
     Amplification of the target nucleic acid regions. A pool of 196 amplicon primer pairs in a concentration of 50 nM for each primer were added to a single PCR tube, 50 ng of human genomic DNA (GenBank No.: NA12878) and 10 μL of amplification reaction mixture which contains 3% glycerol, 0.2 nM dNTPs, 50 nM pyrophosphate and 2 units of KlenTaq-S DNA polymerase to a final volume of 20 μL with DNase/RNase free water. The PCR tube was put on a thermal cycler and run the following temperature profile to get the amplified amplicon library. An initial holding stage was carried out at 98° C. for 2 minutes, followed by 98° C. 15 seconds, 55° C. 8 minutes, for 17 cycles. After cycling, the reaction was held at 72° C. for 5 minutes and then 4° C. until proceeding to the beads purification step to remove excess primers. The tube cap was carefully removed and 24 μL of Agencourt AMPure® XP Reagent (Beckman Coulter, CA) was added to the reaction mixture to purify the DNA. The reaction mixture was vortex mixed and incubated for 5 minutes at room temperature. The tube was placed in a magnetic rack and incubated until solution clears. The supernatant was carefully remove and discarded without disturbing the pellet, then 150 μL of freshly prepared 70% ethanol was added. The reaction mixture was vortexed to mix well, pulsed spin, and incubated until solution clears. The supernatant was carefully removed and discarded without disturbing the pellet. Another portion of 150 μL of freshly prepared 70% ethanol was added. The reaction mixture was vortexed to mix well, pulsed spin, and incubated until solution clears. The supernatant was carefully removed and discarded without disturbing the pellet. Leave the tube open to evaporate for about 2 minutes. The remaining bead pellet in the tube contains the purified DNA. 
     Construction of library. Then 50 μL of Platinum® PCR SuperMix High Fidelity DNA Polymerase (Thermo Fisher, Cat #12532016) and 2 μL of Library Amplification Barcoded Primer Mix (10 μM concentration for each primer was added to bead pellet). The sequence of each primer is shown in Table 4. The PCR tube was put on a thermal cycler and run the following temperature profile to get the amplified amplicon library. An initial holding stage was carried out at 98° C. for 2 minutes, followed by 98° C. 15 seconds, 60° C. 1 minute, for 5 cycles. After cycling, the reaction was held at 72° C. for 5 minutes and then kept at 4° C. until purification. The tube cap was carefully removed and 44 μL of Agencourt AMPure® XP Reagent (Beckman Coulter, CA) was added to the reaction mix for purifying the product. The reaction was vortex mixed and incubated for 5 minutes at room temperature. The tube was placed in a magnetic rack and incubated until solution clears. The supernatant was carefully removed and discarded without disturbing the pellet. Then 150 μL of freshly prepared 70% ethanol was added to the pellet; the reaction mixture was vortexed to mix well, pulsed spin, and incubated until solution clears; the supernatant was carefully removed and discarded without disturbing the pellet; the foregoing wash step was repeated for another time. After the wash, leave the tube open to evaporate for about 2 minutes. Then 50 μL of low TE buffer was added and the solution was vortexed thoroughly. The tube was placed in the magnet until solution clears. The supernatant containing library was collected to a separate clean tube. The library was quantified using Qubit® 2.0 Fluorometer (Life Technologies, CA) and Bioanalyzer (Agilent Technologies, CA) according to manufacturer protocol. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Primers for construction of library 
               
            
           
           
               
               
               
            
               
                 Primer Name 
                 SEQ ID NO: 
                 Primer Sequence 
               
               
                   
               
               
                 R2TruSeqBC001 
                 393 
                 CAAGCAGAAGACGGCATACGAGATcgcgactgaaGT 
               
               
                   
                   
                 GACTGGAGTTCAGACGTGT 
               
               
                   
               
               
                 R2TruSeqBC002 
                 394 
                 CAAGCAGAAGACGGCATACGAGATagcatcgataGTG 
               
               
                   
                   
                 ACTGGAGTTCAGACGTGT 
               
               
                   
               
               
                 R2TruSeqBC003 
                 395 
                 CAAGCAGAAGACGGCATACGAGATcgacacatggGT 
               
               
                   
                   
                 GACTGGAGTTCAGACGTGT 
               
               
                   
               
               
                 R2TruSeqBC004 
                 396 
                 CAAGCAGAAGACGGCATACGAGATcgactacgcaGT 
               
               
                   
                   
                 GACTGGAGTTCAGACGTGT 
               
               
                   
               
               
                 R2TruSeqBC005 
                 397 
                 CAAGCAGAAGACGGCATACGAGATcactgctgagGTG 
               
               
                   
                   
                 ACTGGAGTTCAGACGTGT 
               
               
                   
               
               
                 R2TruSeqBC006 
                 398 
                 CAAGCAGAAGACGGCATACGAGATtcgctgtacaGTG 
               
               
                   
                   
                 ACTGGAGTTCAGACGTGT 
               
               
                   
               
               
                 R2TruSeqBC007 
                 399 
                 CAAGCAGAAGACGGCATACGAGATcgctgcagtaGTG 
               
               
                   
                   
                 ACTGGAGTTCAGACGTGT 
               
               
                   
               
               
                 R2TruSeqBC008 
                 400 
                 CAAGCAGAAGACGGCATACGAGATagacttgcagGTG 
               
               
                   
                   
                 ACTGGAGTTCAGACGTGT 
               
               
                   
               
               
                 R1_TruSeq_primer 
                 401 
                 AATGATACGGCGACCACCGAGATCTACACTCTTT 
               
               
                   
                   
                 CCCTACACGAC 
               
               
                   
               
            
           
         
       
     
     The library was sequenced on Illumina MiSeq sequencer according to manufacturer&#39;s procedure. 
     Data Processing 
     Sequencing reads were aligned to GRC37/hg19 reference genome downloaded from web of ucsc genome browser (hgdownload.soe.ucsc.edu/goldenPath/hg19/bigZips/) using the software of bowtie2 (downloaded from sourceforge.net/projects/bowtie-bio/files/bowtie/1.2.1.1) with default settings. The aligned reads were further assigned to amplicons based on the match between positions of reads of R1 and R2 in genome and positions of forward and reverse primers of designed assays. The preliminary results indicated that performances of cancer hot spot panel were (1) 69.7% reads aligned to genome; (2) 95.5% reads aligned to target regions of design; (3) 98.1% of assays with amplicon read coverage within 5-fold of the mean average. 
     Example 2. Multiplex Enrichment of Mutant Nucleic Acid for Sequencing 
     In 20 μL PCR reaction solution, two pools of 8 primer pairs (see Table 5) with each primer containing dideoxynucleotide at its 3′ end in 0.5 μM concentration were added together with 2 μL of 10×PCR buffer, 3 mM MgCl 2 , 0.2 mM dNTP, 50 nM pyrophosphate, 2 units of AmpliTaq DNA polymerase FS and 1%, 0.1% or 0.01% mutant nucleic acid (Horizon discovery, Cambridge, United Kingdomin), 30 ng of human genomic DNA (GenBank No.: NA12878). The PCR tube was loaded on a thermal cycler and run the following temperature profile: 95° C. for 2 min; 95° C. 15 seconds, 65° C. 120 seconds, for 40 cycles; hold at 4° C. 5 μL ExoSAP-IT™ solution (Affymetrix, CA) was added to the tube, and the reaction was incubated at 37° C. for 15 min, 80° C. for 10 min, held at 4° C. 2 μL of reaction solution was used to perform cycle sequencing with BigDye™ Terminator v3.1 cycle sequencing kit (Life Technologies, CA) and the resulting DNA was purified according to manufacturer protocol. The purified sample electrophoresis was carried out on ABI Prism 3730 DNA analyzer according to manufacturer recommended protocol. Electropherogram in  FIG.  12    showed examples of enrichment of mutant nucleic acid (EGFR COSMIC6240 mutation, EGFR COSMIC6252 mutation, COSMIC6241 mutation, COSMIC6224 mutation, COSMIC6223 mutation and COSMIC6213 mutation from  FIG.  12    from 1%, 0.1% or 0.01% (Mol/Mol) of mutant in wildtype background after amplification reaction. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Primers for enrichment of mutant nucleic acid 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 SEQ 
                   
                   
                 SEQ 
                   
               
               
                 Pool 
                 Forward 
                 ID 
                 Forward Primer 
                 Reverse 
                 ID 
                 Reverse Primer 
               
               
                 ID 
                 Primer ID 
                 NO: 
                 Sequence 
                 Primer ID 
                 NO: 
                 Sequence 
               
               
                   
               
               
                 PMS001 
                 SMDM13CF0033 
                 402 
                 CAGGAAACAG 
                 SMDM13MR0033 
                 403 
                 TGTAAAACGA 
               
               
                   
                   
                   
                 CTATGACCGTG 
                   
                   
                 CGGCCAGTCG 
               
               
                   
                   
                   
                 GAGAAGCTCCC 
                   
                   
                 AACGCACCGG 
               
               
                   
                   
                   
                 AACCAAGC 
                   
                   
                 AGCT 
               
               
                   
                 SMDM13MF0055 
                 404 
                 TGTAAAACGAC 
                 SMDM13CR0055 
                 405 
                 CAGGAAACA 
               
               
                   
                   
                   
                 GGCCAGTGAAA 
                   
                   
                 GCTATGACCG 
               
               
                   
                   
                   
                 GTTAAAATTCC 
                   
                   
                 GCCTGAGGTT 
               
               
                   
                   
                   
                 CGTCGCTATCA 
                   
                   
                 CAGAGCCATG 
               
               
                   
                   
                   
                 AA 
                   
                   
                   
               
               
                   
                 SMDM13CF0014 
                 406 
                 CAGGAAACAG 
                 SMDM13MR0014 
                 407 
                 TGTAAAACGA 
               
               
                   
                   
                   
                 CTATGACCGAA 
                   
                   
                 CGGCCAGTGG 
               
               
                   
                   
                   
                 GCCACACTGAC 
                   
                   
                 CACGTGGGGG 
               
               
                   
                   
                   
                 GTGCCTCT 
                   
                   
                 TTGTCCACGA 
               
               
                   
                 SMDM13CF0041 
                 408 
                 CAGGAAACAG 
                 SMDM13MR0041 
                 409 
                 TGTAAAACGA 
               
               
                   
                   
                   
                 CTATGACCCAG 
                   
                   
                 CGGCCAGTGC 
               
               
                   
                   
                   
                 CCAGGAACGTA 
                   
                   
                 ACCCAGCAGT 
               
               
                   
                   
                   
                 CTGGTGAA 
                   
                   
                 TTGGCCC 
               
               
                   
               
               
                 PMS002 
                 SMDM13CF0002 
                 410 
                 CAGGAAACAG 
                 SMDM13MR0002 
                 411 
                 TGTAAAACGA 
               
               
                   
                   
                   
                 CTATGACCGTG 
                   
                   
                 CGGCCAGTTG 
               
               
                   
                   
                   
                 GAGAAGCTCCC 
                   
                   
                 CCGAACGCAC 
               
               
                   
                   
                   
                 AACCAAGC 
                   
                   
                 CGGAGCA 
               
               
                   
                 SMDM13MF0010 
                 412 
                 TGTAAAACGAC 
                 SMDM13CR0010 
                 413 
                 CAGGAAACA 
               
               
                   
                   
                   
                 GGCCAGTGAAA 
                   
                   
                 GCTATGACCG 
               
               
                   
                   
                   
                 GTTAAAATTCC 
                   
                   
                 GCCTGAGGTT 
               
               
                   
                   
                   
                 CGTCGCTATCA 
                   
                   
                 CAGAGCCATG 
               
               
                   
                   
                   
                 AGA 
                   
                   
                   
               
               
                   
                 SMDM13MF0044 
                 414 
                 TGTAAAACGAC 
                 SMDM13CR0044 
                 415 
                 CAGGAAACA 
               
               
                   
                   
                   
                 GGCCAGTCACC 
                   
                   
                 GCTATGACCG 
               
               
                   
                   
                   
                 GTGCAGCTCAT 
                   
                   
                 TTGAGCAGGT 
               
               
                   
                   
                   
                 CAT 
                   
                   
                 ACTGGGAGCC 
               
               
                   
                   
                   
                   
                   
                   
                 A 
               
               
                   
                 SMDM13CF0009 
                 416 
                 CAGGAAACAG 
                 SMDM13MR0009 
                 417 
                 TGTAAAACGA 
               
               
                   
                   
                   
                 CTATGACCCAG 
                   
                   
                 CGGCCAGTCT 
               
               
                   
                   
                   
                 CCAGGAACGTA 
                   
                   
                 TTCTCTTCCG 
               
               
                   
                   
                   
                 CTGGTGAA 
                   
                   
                 CACCCAGCT 
               
               
                   
               
            
           
         
       
     
     Example 3. Enrichment of Mutant Nucleic Acid by Mismatched PAP Primers for Sequencing 
     In 20 μL PCR reaction solution, a pair of primers (one primer is SMDCR0166 and the other primer is selected from one of SMDMF0166, SMDMF0166G3, SMDMF0166G6, SMDMF0166C9, SMDMF0166C12, SMDMF0166G15) (see Table 6) with each primer containing dideoxynucleotide at its 3′ end in 0.5 μM concentration were added with 2 μL of 10×PCR buffer, with final concentration of 3 mM MgCl 2 , 0.2 mM dNTP, 90 μM of pyrophosphate and 2 units of KlenTaq-S. 30 ng of 100% wild type human genomic DNA (see Table 6) or wild type human genomic DNA spiked with 0.1% mutant genomic DNA (EGFR T790M, see Table 6) was also added to the PCR reaction mixture. The PCR tube was loaded on a thermal cycler and run the following temperature profile: 95° C. for 2 min; 95° C. 15 seconds, 65° C. 120 seconds, for 40 cycles; held at 4° C. 5 μL ExoSAP-IT™ solution (Affymetrix, CA) was added to the tube, and the reaction was incubated at 37° C. for 15 min, 80° C. for 10 min, held at 4° C. 2 μL of treated reaction solution was used to perform cycle sequencing reaction with BigDye™ Terminator v3.1 cycle sequencing kit (Life Technologies, CA) and purified according to manufacturer protocol. The purified sample electrophoresis was carried out on ABI Prism 3730 DNA analyzer according to manufacturer recommended protocol. The results are shown in  FIG.  13   . It can be seen that the mismatched nucleotide contributes to decreasing false positive results. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Primer and template sequence for EGFR T790M detection 
               
            
           
           
               
               
               
            
               
                 Primer or Template 
                 SEQ ID NO: 
                 Sequence 
               
               
                   
               
               
                 SMDMF0166 
                 418 
                 CTCCACCGTGCAGCTCATCAddT 
               
               
                   
               
               
                 SMDMF0166G3 
                 419 
                 CTCCACCGTGCAGCTCATGAddT 
               
               
                   
               
               
                 SMDMF0166G6 
                 420 
                 CTCCACCGTGCAGCTGATCAddT 
               
               
                   
               
               
                 SMDMF0166C9 
                 421 
                 CTCCACCGTGCAGCTCATCAddT 
               
               
                   
               
               
                 SMDMF0166C12 
                 422 
                 CTCCACCGTGCAGCTCATCAddT 
               
               
                   
               
               
                 SMDMF0166015 
                 423 
                 CTCCACGGTGCAGCTCATCAddT 
               
               
                   
               
               
                 SMDCR0166 
                 424 
                 GTTGAGCAGGTACTGGGAGCCddA 
               
               
                   
               
               
                 WT Template 
                 425 
                 GAGGTGGCACGTCGAGTAGTGCGTCGAGTACG 
               
               
                 (3′ to 5′) 
                   
                 GGAAGCCGACGGAGGACCTGATACAGGC--- 
               
               
                   
               
               
                 Mut Template 
                 426 
                 GAGGTGGCACGTCGAGTAGTACGTCGAGTACGG 
               
               
                 (3′ to 5′) 
                   
                 GAAGCCGACGGAGGACCTGATACAGGC--- 
               
               
                   
               
            
           
         
       
     
     Example 4. Enrichment of Mutant Nucleic Acid by PAP Primers and Proof-Reading PFU Enzyme for Sequencing 
     In 20 μL PCR reaction solution, forward and reverse primer pairs (see Table 7) with each primer containing dideoxynucleotide at its 3′ end in 0.5 μM concentration were added with 2 μL of 10×PCR buffer, with final concentration of 3 mM MgCl 2 , 0.2 mM dNTP, 90 μM of pyrophosphate and 2 units of KlenTaq-S with or without 2 units of Pfu DNA polymerase (Promega, Wis.) were added to the reaction mixture. Then 30 ng of 100% wild type human genomic DNA (NA12878) (see Table 7) or wild type human genomic DNA (NA12878) spiked with 0.10% mutant genomic DNA (EGFR G719S, see Table 7) was also added to the PCR reaction mixture. The PCR tube was loaded on a thermal cycler and run the following temperature profile: 95° C. for 2 min; 95° C. 15 seconds, 65° C. 120 seconds, for 40 cycles, held at V° C. 5 μL ExoSAP-IT™ solution (Affymetrix, CA) was added to the tube, and the reaction was incubated at 37° C. for 15 min, 80° C. for 10 min, held at 4° C. 2 μL of treated reaction solution was used to perform cycle sequencing with BigDye™ Terminator v3.1 cycle sequencing kit (Life Technologies, CA) and purified according to manufacturer protocol. The purified sample electrophoresis was carried out on ABI Prism 3730 DNA analyzer according to manufacturer recommended protocol. The results are shown in  FIG.  14   . It can be seen that the proof-reading PFU enzyme contributes to decreasing false positive results. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Primer and template sequence for EGFR G719S detection 
               
            
           
           
               
               
               
            
               
                 Primer or 
                 SEQ 
                   
               
               
                 Template 
                 ID NO: 
                 Sequence 
               
               
                   
               
               
                 SMDMF0166 
                 427 
                 CTCCACCGTGCAGCTCATCAddT 
               
               
                   
               
               
                 SMDMF0166G3 
                 428 
                 CTCCACCGTGCAGCTCATGAddT 
               
               
                   
               
               
                 SMDMF0166G6 
                 429 
                 CTCCACCGTGCAGCTGATCAddT 
               
               
                   
               
               
                 SMDMF0166C9 
                 430 
                 CTCCACCGTGCAGCTCATCAddT 
               
               
                   
               
               
                 SMDMF0166C12 
                 431 
                 CTCCACCGTCCAGCTCATCAddT 
               
               
                   
               
               
                 SMDMF0166015 
                 432 
                 CTCCACCGTGCAGCTCATCAddT 
               
               
                   
               
               
                 SMDCR0166 
                 433 
                 GTTGAGCAGGTACTGGGAGCCddA 
               
               
                   
               
               
                 WT Template 
                 434 
                 GAGGTGGCACGTCGAGTAGTGCGTCGAGTACGGGAA 
               
               
                 (3′ to 5′) 
                   
                 GCCGACGGAGGACCTGATACAGGC--- 
               
               
                   
               
               
                 Mut Template 
                 435 
                 GAGGTGGCACGTCGAGTAGTACGTCGAGTACGGGAA 
               
               
                 (3′ to 5′) 
                   
                 GCCGACGGAGGACCTGATACAGGC ---