Patent Publication Number: US-2019177786-A1

Title: Methods and materials for the effective use of combined targeted enrichment of genomic regions and low coverage whole genome sequencing

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims priority to U.S. Application Ser. No. 62/351,742, filed on Jun. 17, 2016. The disclosure of the prior application is considered part of the disclosure of this application, and is incorporated in its entirety into this application. 
    
    
     BACKGROUND 
     1. Technical Field 
     This document relates to methods and materials involved in using low coverage whole genome sequencing (LC-WGS) techniques to assess genomes. For example, this document provides methods and materials for performing targeted enrichment of genomic regions (e.g., targeted amplification and/or targeted capture techniques) in combination with LC-WGS techniques to assess genomes. 
     2. Background Information 
     High coverage whole genome sequencing techniques, which could theoretically be used to call variants, amplifications, and deletions genome wide, is currently not used in clinical applications due to the high cost of the test as well as the complexity of interpreting results. One whole genome sequencing assay used for clinical application is the LC-WGS assay that has a coverage of about 1× or less. LC-WGS was used successfully for the non-invasive screening of fetuses to report trisomy of chromosome 13, 18, and 21. 
     SUMMARY 
     This document provides methods and materials for using low coverage whole genome sequencing techniques to assess genomes. For example, this document provides methods and materials for using targeted nucleic acid amplification and/or targeted nucleic acid capture techniques in combination with low coverage whole genome sequencing techniques to obtain high coverage sequencing data for one or more pre-selected regions of a genome. Generally, during whole genome sequencing, DNA is fragmented into short fragments that are about 400 to 500 base pairs long. About 100 to 150 base pairs are sequenced at one or both ends of these fragments. A sequenced section of a DNA fragment is called a sequence read. Coverage refers to the number of reads spanning over a specific genomic location. A sample sequenced at 10× average coverage means that, on average, 10 reads span the genomic regions that were sequenced. 
     As described herein, combining targeted nucleic acid amplification and/or targeted nucleic acid capture techniques with low coverage whole genome sequencing techniques can generate a sequencing coverage that is less than about 1× for the regions of the genome outside the one or more pre-selected regions amplified and/or captured and a sequencing coverage that is greater than about 500× for the one or more pre-selected regions. For example, combining targeted nucleic acid amplification and/or targeted nucleic acid capture techniques with low coverage whole genome sequencing can provide a composite low resolution view of genomic variations across the genome with a high resolution view of genomic variations in one or more selected regions that were enriched via nucleic acid amplification and/or nucleic acid capture techniques. This can allow clinicians to obtain high coverage sequencing data for one or more pre-selected regions of a genome while performing cost effective, low coverage whole genome sequencing. 
     In general, one aspect of this document features a method for increasing the number of sequencing reads of one or more pre-selected genomic regions using low coverage whole genome sequencing. The method comprises, or consist essentially of, performing an amplification reaction using a genomic nucleic acid sample to amplify one or more pre-selected genomic regions, thereby forming an amplified sample, and performing low coverage whole genome sequencing using the amplified sample, wherein the coverage of the pre-selected genomic regions using the low coverage whole genome sequencing is greater than 250×, and wherein the coverage of regions outside the pre-selected genomic regions using the low coverage whole genome sequencing is less than 10×, less than 5×, or less than 3×. The one or more pre-selected genomic regions can be from one pre-selected genomic region to 2500 pre-selected genomic regions. The one or more pre-selected genomic regions can be from one pre-selected genomic region to 2000 pre-selected genomic regions. The one or more pre-selected genomic regions can be from one pre-selected genomic region to 1500 pre-selected genomic regions. The low coverage whole genome sequencing can be whole genome sequencing with less than 2× genome wide coverage. The low coverage whole genome sequencing can be whole genome sequencing with less than 1× genome wide coverage. The genomic nucleic acid sample can be a human genomic nucleic acid sample. The coverage of the pre-selected genomic regions using the low coverage whole genome sequencing can be greater than 500×. The coverage of the pre-selected genomic regions using the low coverage whole genome sequencing can be greater than 1000× (or greater than 1500×, greater than 2000×, greater than 3000×, greater than 5000×, greater than 7500×, or greater than 10000×). The method can comprise performing the amplification reaction using the genomic nucleic acid sample to amplify one or more pre-selected genomic regions having a length from about 150 bp to about 750 bp. 
     In another aspect, this document features a method for increasing the number of sequencing reads of one or more pre-selected genomic regions using low coverage whole genome sequencing. The method comprises, or consists essentially of, performing a nucleic acid capture reaction using a genomic nucleic acid sample to enrich one or more pre-selected genomic regions, thereby forming an enriched sample, and performing low coverage whole genome sequencing using the enriched sample, wherein the coverage of the pre-selected genomic regions using the low coverage whole genome sequencing is greater than 250×, and wherein the coverage of regions outside the pre-selected genomic regions using the low coverage whole genome sequencing is less than 10×, less than 5×, or less than 3×. The one or more pre-selected genomic regions can be from one pre-selected genomic region to 2500 pre-selected genomic regions. The one or more pre-selected genomic regions can be from one pre-selected genomic region to 2000 pre-selected genomic regions. The one or more pre-selected genomic regions can be from one pre-selected genomic region to 1500 pre-selected genomic regions. The low coverage whole genome sequencing can be whole genome sequencing with less than 2× genome wide coverage. The low coverage whole genome sequencing can be whole genome sequencing with less than 1× genome wide coverage. The genomic nucleic acid sample can be a human genomic nucleic acid sample. The coverage of the pre-selected genomic regions using the low coverage whole genome sequencing can be greater than 500×. The coverage of the pre-selected genomic regions using the low coverage whole genome sequencing can be greater than 1000× (or greater than 1500×, greater than 2000×, greater than 3000×, greater than 5000×, greater than 7500×, or greater than 10000×). The method can comprise performing the nucleic acid capture reaction using the genomic nucleic acid sample to capture one or more pre-selected genomic regions having a length from about 150 bp to about 750 bp. 
     Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. 
     The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. 
    
    
     
       DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a schematic of the steps of an exemplary workflow for the processing of a sequencing protocol according to one embodiment. 
         FIG. 2  is a graph plotting LC-WGS sequencing coverage of a normal sample. The X axis displays the coverage on each chromosome that are numbered in ascending order. The Y axis is the number of reads mapped to the genomic region associated to a bin. Bioinformatics techniques are applied to the data to optimize evenness of coverage across the genome (X axis). Each dot on the plot represents a bin of 10 kb. In this example, bins include in average 50 reads, but fluctuate between 10× and 80×. In this sample, no statistically significant amplifications or deletions are observed. 
         FIG. 3  is a graph plotting the sequencing results obtained by combining the use of low coverage whole genome sequencing and amplification of selected regions. The X axis displays the coverage on each chromosome that are numbered in ascending order. The Y axis is the number of reads divided by 1000 that are mapped to the genomic region associated to a bin. The sample sequenced is a normal sample. Each circle represents a bin of 10 kb. The LC-WGS is represented by set of grey circles that form a base line due to the scale of the plot. On average, 50 reads of 150 bp are found in each bin. The black circles represent the coverage level of 97 loci that are 90 bases long and that were amplified using a PCR assay (amplicon). The coverage of these loci can reach for some of them 100,000× and therefore can be used to call genotypes, identify somatic mutations, identify breakpoints associated to structural variants or identify change of coverage informative of the amplification or deletion of these regions. In this example, the amplified regions overlap with SNPs from which the genotypes can be called accurately. The genotypes of SNPs cannot be called from low coverage sequencing alone. 
         FIGS. 4A and 4B . LC-WGS coverage computed from the reads extracted from a targeted amplification assay where PCR amplification was performed below saturation. No-coverage regions correspond to centromers. Chromosome wide amplification, local and complex patterns of amplification are clearly visible in these plots. 
     
    
    
     DETAILED DESCRIPTION 
     This document provides methods and materials for using low coverage whole genome sequencing techniques to assess genomes (e.g., genomic variations). For example, this document provides methods and materials for using targeted nucleic acid amplification and/or targeted nucleic acid capture techniques in combination with low coverage whole genome sequencing techniques to obtain high coverage sequencing data (e.g., over 500× coverage) for one or more selected regions of a genome. 
     Low coverage whole genome sequencing can be performed by limiting the concentration of DNA input in the sequencing reaction. A sample from a healthy human and assessed using low coverage whole genome sequencing without enriching pre-selected regions can be as shown in  FIG. 2 . In some cases, samples can be multiplexed in a single whole genome sequencing assay. The concentration of each sample can be controlled to ensure that the DNA concentration is proportional to the number of samples. For example, the Illumina HiSeq 2000 can be set to produce per lane of flow cell: 300,000,000 reads that are 100 base pair long. Since the human genome is about 3 billion bases long, the whole genome of a single sample could be sequenced with a coverage of 10× coverage. If 10 samples are sequenced together in a flow cell lane, then the coverage per sample will be on average about 1×. 
     As described herein, combining targeted nucleic acid amplification and/or capture techniques with low coverage whole genome sequencing techniques can generate a sequencing coverage that is from less than about 1× coverage for the regions of the genome outside the one or more selected regions amplified and/or captured and a sequencing coverage that can be greater than 50,000× for the one or more selected regions (see, e.g.,  FIG. 3 ). 
     Any appropriate nucleic acid amplification technique can be used to increase the sequence read coverage of one or more selected regions targeted for amplification. For example, PCR amplification can be used to increase the sequence read coverage of one or more selected regions when low coverage whole genome sequencing is used. In some cases, nucleic acid amplification techniques can be used to amplify more than 2000 regions of a genome. Increasing the number of amplified regions decreases the number of reads available to cover the whole genome and therefore decreases the LC-WGS coverage. 
     In some cases, nucleic acid capture techniques can be used in addition to, or in place of, nucleic acid amplification techniques to increase the sequence read coverage of one or more selected regions targeted for enrichment. Any appropriate nucleic acid capture technique can be used to increase the sequence read coverage of one or more selected regions targeted for enrichment. For example, DNA can be used as bait to capture a targeted sequence as described elsewhere (Hagemann et al.,  Cancer Genetics,  206:420-431 (2014)). 
     In some cases, in a single experimental protocol, a low coverage whole genome sequencing assay can be combined with a targeted amplicon assay, where PCR is used to amplify selected regions of the genome. In some cases, the amplification step can be replaced with a nucleic acid capture technique to capture genomic regions that can be combined with a low coverage whole genome sequencing assay. The sequencing result can be a combination of low coverage whole genome sequencing that provides an overview of the genomic amplification/deletion (e.g., duplications or other genomic amplifications or genomic deletions) landscape of the genome with high coverage sequencing data for the amplified and/or captured regions (e.g., a coverage up to several 1000×; see, e.g.,  FIG. 3 ). This high coverage sequencing data obtained using an otherwise low coverage whole genome sequencing assay can be used to identify single nucleotide variants, indels, translocations, and/or copy number changes at a high sensitivity. For example, selected genomic regions can be selected and enriched (e.g., amplified) so that high coverage is obtained for those regions to identify SNPs, genomic amplifications, genomic deletions, and translocations. In some cases, the high sensitivity in these regions can be set to be similar to that obtained using fluorescence in situ hybridization (FISH) techniques. 
     Briefly, one exemplary implementation of the methods provided herein can include the following steps: (a) DNA extraction, (b) an optional whole genome amplification step if enough DNA is not available, (c) PCR amplification of one or more targeted genomic regions with a controlled number of PCR cycles, (d) optional genomic barcoding if multiple samples are to be sequenced in a single experiment, and (e) low coverage whole genome sequencing. Other exemplary implementations of the methods provided herein can be carried out as set forth in  FIG. 1 . 
     Since the amount of DNA sequenced is about constant per sequencing experiment, the number and length of the genomic regions to be amplified, the coverage level expected for these regions, and the number of samples to be sequenced in a single experiment can be directly related to the sequencing reads left to cover the whole genome. 
     The following parameters can be used to design an assay provided herein such that it achieves a particular coverage for the genomic regions enriched and those genomic regions not enriched: (a) total number of reads produced by the sequencing platform, (b) number of samples to sequence in a single experiment, (c) number of target regions to amplify or capture, (d) length of the region to amplified or captured, and (e) expected coverage of the enriched target regions. 
     The following defines the relationship between these parameters: 
       LC=(RS*RL−AN*AL*AC)/LG
 
     where: 
     RS is the number of sequenced read per sample 
     RL is the length of a read (in bases) 
     AN is the number of amplicons 
     AL is the length of the amplicons (in bases) 
     AC is the coverage of each amplicons 
     LC is the coverge of the LC-WGS 
     LG is the number of base pair in the sequenced genome 
     Table 1 sets forth different exemplary combinations of the parameters RL, AN, AL, AC, and LC. LG is set to 3 billion base pairs (human genome) 
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 reads per 
                 read 
                 number of 
                 amplicon 
                 amplicon 
                 LC-WGS 
               
               
                 sample 
                 length 
                 amplicons 
                 length 
                 coverage 
                 coverage 
               
               
                 (RS) 
                 (RL) 
                 (AN) 
                 (AL) 
                 (AC) 
                 (LC) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 30,000,000 
                 100 
                 100 
                 500 
                 5,000 
                 0.92 
               
               
                 30,000,000 
                 100 
                 100 
                 1000 
                 5,000 
                 0.83 
               
               
                 30,000,000 
                 150 
                 200 
                 500 
                 5,000 
                 1.33 
               
               
                 30,000,000 
                 150 
                 200 
                 1000 
                 5,000 
                 1.17 
               
               
                 40,000,000 
                 150 
                 200 
                 500 
                 10,000 
                 1.67 
               
               
                 40,000,000 
                 150 
                 200 
                 1000 
                 10,000 
                 1.33 
               
               
                 40,000,000 
                 150 
                 300 
                 500 
                 10,000 
                 1.50 
               
               
                 40,000,000 
                 150 
                 300 
                 1000 
                 10,000 
                 1.00 
               
               
                   
               
            
           
         
       
     
     In some cases, the methods and materials provided herein can be used for the early detection of cancer or to stratify tumors on the basis of, for example, genome wide aneuploidy events and, in the target enriched regions: copy number alterations, mutations, and diverse structural variants. In some cases, the methods and materials provided herein can be used to monitor recurrence of cancer following treatment (e.g., surgery) with the enriched (e.g., amplified and/or captured) selected regions being selected based on the SNPs or translocations of the original tumor. 
     Any appropriate genome can be assessed using the methods and materials provided herein. For example, the genome of a human, horse, bovine species, dog, cat, or monkey can be assessed using the methods and materials provided herein. In addition, any appropriate sample containing genomic nucleic acid can be used as described herein. For example, the methods and materials provided herein can be used to analyze DNA extracted from cells or cell-free DNA extracted from blood, from brushings, or tampons. In some cases, the methods and materials described herein can be used to assess nucleic acid from fresh samples, frozen samples, or formalin-fixed paraffin embedded samples. Any appropriate sample preparation technique can be used to extract DNA from cells or extract cell-free DNA from blood, feces, urine, tampons, or brushing samples. For example, a nucleic acid extraction kits can be used. 
     Any appropriate genome region can be a selected target region that is amplified or enriched to increase its sequence read coverage during low coverage whole genome sequencing. For example, any one or more of the nucleic acid regions set forth in Table 2 (or portions thereof) can be amplified as described herein to generate amplified selected regions that provide an increased sequence read coverage during low coverage whole genome sequencing. Such nucleic acid regions can be used to detect a genetic defect or element within the amplified regions. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Exemplary selected regions of human genome 
               
               
                 for amplification or capture enrichment. 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 SEQ 
                   
                 SEQ 
                   
               
               
                   
                   
                   
                 Exon 
                 Exon 
                 Primer 
                 Primer 
                   
                   
                 ID 
                   
                 ID 
                   
               
               
                 Gene 
                 Chr 
                 Exon 
                 Start 
                 End 
                 Start 
                 End 
                 Len 
                 Fwd Primer 
                 NO: 
                 Rev Primer 
                 NO: 
                 ID 
               
               
                   
               
               
                 CCND1 
                 chr11 
                  1 
                  69455872 
                  69456279 
                  69455842 
                  69456390 
                 549 
                 GGCTTTGATCTTTGCTTAAC 
                   9 
                 AAACTTCAAAGTTCTAGCGG 
                 162 
                   1 
               
               
                   
               
               
                 CCND1 
                 chr11 
                  2 
                  69457798 
                  69458014 
                  69457592 
                  69458125 
                 534 
                 GGACTTTCCCTTTCAGTTTC 
                  10 
                 AGGAGCAGATATGTCAGAGG 
                 163 
                   2 
               
               
                   
               
               
                 CCND1 
                 chr11 
                  3 
                  69458599 
                  69458759 
                  69458336 
                  69458863 
                 528 
                 GGAGGTCTTTTTGTTTCCAC 
                  11 
                 GACATCTTCCCAGACAGCAC 
                 164 
                   3 
               
               
                   
               
               
                 CCND1 
                 chr11 
                  4 
                  69462761 
                  69462910 
                  69462512 
                  69463092 
                 581 
                 TTCCTTGGTTATGTTTGAGTC 
                  12 
                 TCTAGGAGCAGTGGAAGAAG 
                 165 
                   4 
               
               
                   
               
               
                 CCND1 
                 chr11 
                  5 
                  69465885 
                  69469242 
                  69465779 
                  69466337 
                 559 
                 TTGCTCTTATAAAGGCTTCC 
                  13 
                 TATCATCTGTAGCACAACCC 
                 166 
                   5 
               
               
                   
               
               
                 CCND1 
                 chr11 
                  5 
                  69465885 
                  69469242 
                  69466159 
                  69466730 
                 572 
                 AAGCTTCATTCTCCTTGTTG 
                  14 
                 ACGCTACTGTAACCAAGAGG 
                 167 
                   6 
               
               
                   
               
               
                 CCND1 
                 chr11 
                  5 
                  69465885 
                  69469242 
                  69466597 
                  69467101 
                 505 
                 GCATCTCTGTACTTTGCTTG 
                  15 
                 AACAGCGCTATTTCCTACAC 
                 168 
                   7 
               
               
                   
               
               
                 CCND1 
                 chr11 
                  5 
                  69465885 
                  69469242 
                  69467056 
                  69467580 
                 525 
                 ATTTCCAAGCACTTTCAGTC 
                  16 
                 AGAAGGTTTGTGTGTGTGTG 
                 169 
                   8 
               
               
                   
               
               
                 CCND1 
                 chr11 
                  5 
                  69465885 
                  69469242 
                  69467560 
                  69468087 
                 528 
                 ACACACACACACAAACCTTC 
                  17 
                 CAGCAAACAATGTGAAAGAG 
                 170 
                   9 
               
               
                   
               
               
                 CCND1 
                 chr11 
                  5 
                  69465885 
                  69469242 
                  69468041 
                  69468490 
                 450 
                 GGAAATATTCACATCGCTTC 
                  18 
                 ACTACTATGATGCTACGCCC 
                 171 
                  10 
               
               
                   
               
               
                 CCND1 
                 chr11 
                  5 
                  69465885 
                  69469242 
                  69468254 
                  69468737 
                 484 
                 TGTTTCACAATACCTCATGC 
                  19 
                 GATTTGGAGTCTCTTTAAATTAGC 
                 172 
                  14 
               
               
                   
               
               
                 CCND1 
                 chr11 
                  5 
                  69465885 
                  69469242 
                  69468591 
                  69469036 
                 446 
                 ACCTGTAGGACTCTCATTCG 
                  20 
                 TCTCGATACACACAACATCC 
                 173 
                  13 
               
               
                   
               
               
                 CCND1 
                 chr11 
                  5 
                  69465885 
                  69469242 
                  69469013 
                  69469596 
                 584 
                 TCCTGGATGTTGTGTGTATC 
                  21 
                 AGCCTGCAAATTATTCTCTG 
                 174 
                  12 
               
               
                   
               
               
                 LMO1 
                 chr11 
                  1 
                   8289973 
                   8290182 
                   8289734 
                   8290333 
                 600 
                 GAGACTTCCTAATCCCGCCG 
                  22 
                 CTCTGCTGAGGCGAGTACGG 
                 175 
                  11 
               
               
                   
               
               
                 LMO1 
                 chr11 
                  2 
                   8251837 
                   8252051 
                   8251723 
                   8252126 
                 404 
                 GAGAGGACACACAGGGTACT 
                  23 
                 ATTCTTGGGGGATATTCCTT 
                 176 
                  15 
               
               
                   
               
               
                 LMO1 
                 chr11 
                  3 
                   8248521 
                   8248647 
                   8248278 
                   8248787 
                 510 
                 TATTCACACAGAAATGTGCC 
                  24 
                 TCTTATCCTATTGCCTGAGC 
                 177 
                  16 
               
               
                   
               
               
                 LMO1 
                 chr11 
                  4 
                   8245850 
                   8246268 
                   8245819 
                   8246368 
                 550 
                 AGGTCTGTGTCAGTCATGTG 
                  25 
                 ACATAGCTCACCTCATAGGC 
                 178 
                  17 
               
               
                   
               
               
                 MDM2 
                 chr12 
                  1 
                  69201951 
                  69202271 
                  69201702 
                  69202276 
                 575 
                 GGCTAAAGGAGTGTCACAGC 
                  26 
                 AGTACCTGCTCCTCACCATC 
                 179 
                  18 
               
               
                   
               
               
                 MDM2 
                 chr12 
                  2 
                  69202987 
                  69203072 
                  69202745 
                  69203311 
                 567 
                 AAGTCCTGACTTGTCTCCAG 
                  27 
                 CACGCTTAACAATGTAATGG 
                 180 
                  19 
               
               
                   
               
               
                 MDM2 
                 chr12 
                  3 
                  69207333 
                  69207408 
                  69207149 
                  69207681 
                 533 
                 TGGATTGGATACTGTCTGTG 
                  28 
                 ATTCTGGGAAGGAGTCTACC 
                 181 
                  20 
               
               
                   
               
               
                 MDM2 
                 chr12 
                  4 
                  69210591 
                  69210725 
                  69210331 
                  69210882 
                 552 
                 TTAGTAGAGATGGGACCAGG 
                  29 
                 GGTTCTCAAATAATATGCCG 
                 182 
                  21 
               
               
                   
               
               
                 MDM2 
                 chr12 
                  5 
                  69214104 
                  69214154 
                  69213983 
                  69214509 
                 527 
                 TTTGAATGTGTGCAGTAGTTC 
                  30 
                 TCCTTACACATGGTCCTACC 
                 183 
                  22 
               
               
                   
               
               
                 MDM2 
                 chr12 
                  6 
                  69218142 
                  69218210 
                  69218039 
                  69218363 
                 325 
                 AAATTGCATAAGGGTTTGTG 
                  31 
                 TTCTCTTCCTGAAGCTCTTG 
                 184 
                  23 
               
               
                   
               
               
                 MDM2 
                 chr12 
                  7 
                  69218334 
                  69218431 
                  69218161 
                  69218640 
                 480 
                 CATCTGTGAGTGAGAACAGG 
                  32 
                 GTAAACTGTGCCTGCTGTAG 
                 185 
                  24 
               
               
                   
               
               
                 MDM2 
                 chr12 
                  8 
                  69222550 
                  69222711 
                  69222304 
                  69222899 
                 596 
                 AGATTGTGCCTCTGTACTCC 
                  33 
                 ATTTCTCACAATACCTTGGG 
                 186 
                  25 
               
               
                   
               
               
                 MDM2 
                 chr12 
                  9 
                  69229608 
                  69229764 
                  69229556 
                  69230130 
                 575 
                 ACAGAGGTCAAGAGGTGATG 
                  34 
                 TGGGAAACAGATCTCTAAGG 
                 187 
                  26 
               
               
                   
               
               
                 MDM2 
                 chr12 
                 10 
                  69230451 
                  69230529 
                  69230398 
                  69230878 
                 481 
                 TCTGATTGAAGGAAATAGGG 
                  35 
                 GCCTGTAATTCCAGCTACTC 
                 188 
                  27 
               
               
                   
               
               
                 MDM2 
                 chr12 
                 11 
                  69233053 
                  69239324 
                  69232933 
                  69233478 
                 546 
                 AAACACTGAATATTGAGCCC 
                  36 
                 TGACAAATCACACAAGGTTC 
                 189 
                  28 
               
               
                   
               
               
                 MDM2 
                 chr12 
                 11 
                  69233053 
                  69239324 
                  69233263 
                  69233839 
                 577 
                 CAGAGAGTCATGTGTTGAGG 
                  37 
                 AGTTGGTGTAAAGGATGAGC 
                 190 
                  29 
               
               
                   
               
               
                 MDM2 
                 chr12 
                 11 
                  69233053 
                  69239324 
                  69233819 
                  69234364 
                 546 
                 AGCTCATCCTTTACACCAAC 
                  38 
                 GCTAGATCATGACACTGCAC 
                 191 
                  30 
               
               
                   
               
               
                 MDM2 
                 chr12 
                 11 
                  69233053 
                  69239324 
                  69234347 
                  69234878 
                 532 
                 GCAGTGTCATGATCTAGCAG 
                  39 
                 TGAGGTGAGTAGATCACTTGAG 
                 192 
                  31 
               
               
                   
               
               
                 MDM2 
                 chr12 
                 11 
                  69233053 
                  69239324 
                  69234715 
                  69235284 
                 570 
                 TCTGGGTTCAAGCTATTCTC 
                  40 
                 TTTGTCTTACGGGTAAATGG 
                 193 
                  32 
               
               
                   
               
               
                 MDM2 
                 chr12 
                 11 
                  69233053 
                  69239324 
                  69235142 
                  69235665 
                 524 
                 GCTAAGTAGGATTACAGGCG 
                  41 
                 GCTTGAGAGGAAGTCAAGAG 
                 194 
                  33 
               
               
                   
               
               
                 MDM2 
                 chr12 
                 11 
                  69233053 
                  69239324 
                  69235413 
                  69235862 
                 450 
                 TAAAGTACCTTCTTGGCCTG 
                  42 
                 ACAGAATGCTTTAGTCCACC 
                 195 
                  34 
               
               
                   
               
               
                 MDM2 
                 chr12 
                 11 
                  69233053 
                  69239324 
                  69235711 
                  69236286 
                 576 
                 GTGTTAGTTTCTTTGGGACC 
                  43 
                 GTAATCACCTTTCATCGGAG 
                 196 
                  35 
               
               
                   
               
               
                 MDM2 
                 chr12 
                 11 
                  69233053 
                  69239324 
                  69236212 
                  69236802 
                 591 
                 CTCCTTTGGAGACTTAGAACC 
                  44 
                 AGCTTGTTCTACCAGGAATG 
                 197 
                  36 
               
               
                   
               
               
                 MDM2 
                 chr12 
                 11 
                  69233053 
                  69239324 
                  69236522 
                  69237080 
                 559 
                 AAGGGAGGATATAAGGAACC 
                  45 
                 CTCTCAATAAATGGCCAAAG 
                 198 
                  37 
               
               
                   
               
               
                 MDM2 
                 chr12 
                 11 
                  69233053 
                  69239324 
                  69237017 
                  69237603 
                 587 
                 CCAAATAATGCTTTGAGGAC 
                  46 
                 AAAGAGATTCTGCTTGGTTG 
                 199 
                  38 
               
               
                   
               
               
                 MDM2 
                 chr12 
                 11 
                  69233053 
                  69239324 
                  69237424 
                  69237893 
                 470 
                 GGACTGAGGTAATTCTGCAC 
                  47 
                 CCCATAAACATGTTGAATCC 
                 200 
                  39 
               
               
                   
               
               
                 MDM2 
                 chr12 
                 11 
                  69233053 
                  69239324 
                  69237579 
                  69238177 
                 599 
                 AGCTACAACCAAGCAGAATC 
                  48 
                 TGCAACATCATTCTCTCAAG 
                 201 
                  40 
               
               
                   
               
               
                 MDM2 
                 chr12 
                 11 
                  69233053 
                  69239324 
                  69237775 
                  69238260 
                 486 
                 TTCTGAGGAGTATCGGTAGC 
                  49 
                 ACCATTCACGATCACTTAGG 
                 202 
                  41 
               
               
                   
               
               
                 MDM2 
                 chr12 
                 11 
                  69233053 
                  69239324 
                  69238214 
                  69238663 
                 450 
                 CTTCTCTTAGGTCACATGGC 
                  50 
                 AAGCAGAACCACTTGAACAC 
                 203 
                  42 
               
               
                   
               
               
                 MDM2 
                 chr12 
                 11 
                  69233053 
                  69239324 
                  69238402 
                  69238927 
                 526 
                 TTGTGAGGCACAAATGTAAG 
                  51 
                 TTCACAATGCCATTAACAAC 
                 204 
                  43 
               
               
                   
               
               
                 MDM2 
                 chr12 
                 11 
                  69233053 
                  69239324 
                  69238879 
                  69239450 
                 572 
                 GGTCTGTAGGCTTATGATGG 
                  52 
                 GAGATGTGGGATTGTAGGAC 
                 205 
                  44 
               
               
                   
               
               
                 MDM4 
                 chr1 
                  1 
                 204485506 
                 204485637 
                 204485352 
                 204485901 
                 550 
                 AAATCTGACGACTTTCAACC 
                  53 
                 ACGTCGACTTTAGGTTTGTC 
                 206 
                  45 
               
               
                   
               
               
                 MDM4 
                 chr1 
                  2 
                 204494611 
                 204494724 
                 204494451 
                 204495019 
                 569 
                 AAGATATGCAGAACCTCAGC 
                  54 
                 CATAATTCACTGCAGCTTTG 
                 207 
                  46 
               
               
                   
               
               
                 MDM4 
                 chr1 
                  3 
                 204495487 
                 204495562 
                 204495232 
                 204495823 
                 592 
                 AAATTACCTGGATATGGTGG 
                  55 
                 GTCAGGAGACTGAGACCATC 
                 208 
                  47 
               
               
                   
               
               
                 MDM4 
                 chr1 
                  4 
                 204499811 
                 204499945 
                 204499574 
                 204500079 
                 506 
                 ATCAGTTCATTTCTGTGCTG 
                  56 
                 TGCCTCATAGGCTACCTAAC 
                 209 
                  48 
               
               
                   
               
               
                 MDM4 
                 chr1 
                  5 
                 204501318 
                 204501374 
                 204501252 
                 204501832 
                 581 
                 GGCAAACCACTGATATCTTC 
                  57 
                 GAGACATATCAACCAAAGGC 
                 210 
                  49 
               
               
                   
               
               
                 MDM4 
                 chr1 
                  6 
                 204506557 
                 204506625 
                 204506510 
                 204506840 
                 331 
                 ATGGTTATTACCAGGGAAGG 
                  58 
                 AGAAGTGCTACATCCCAAAG 
                 211 
                  50 
               
               
                   
               
               
                 MDM4 
                 chr1 
                  7 
                 204507336 
                 204507436 
                 204507222 
                 204507638 
                 417 
                 TTCTTGTGTGTAACCCATTG 
                  59 
                 ATCCTAGTACTCACGGGTTG 
                 212 
                  51 
               
               
                   
               
               
                 MDM4 
                 chr1 
                  8 
                 204511911 
                 204512072 
                 204511725 
                 204512265 
                 541 
                 TGAAGTCTAAACAAGGGAGG 
                  60 
                 AACTGAAGTTGGGCATTTAG 
                 213 
                  52 
               
               
                   
               
               
                 MDM4 
                 chr1 
                  9 
                 204513662 
                 204513812 
                 204513529 
                 204514082 
                 554 
                 GTCCACTGAATAAAGGCAAG 
                  61 
                 TACCTTGTTAGCAAAGGGAG 
                 214 
                  53 
               
               
                   
               
               
                 MDM4 
                 chr1 
                 10 
                 204515924 
                 204516005 
                 204515663 
                 204516246 
                 584 
                 TATGGGCATCTTCTCTCTTC 
                  62 
                 CAGAGGCATTTATCTCATCC 
                 215 
                  54 
               
               
                   
               
               
                 MDM4 
                 chr1 
                 11 
                 204518240 
                 204527248 
                 204518078 
                 204518653 
                 576 
                 AAAGACTTTCCTTCATGTGG 
                  63 
                 AAGCTACATGGCTTCAAGAG 
                 216 
                  55 
               
               
                   
               
               
                 MDM4 
                 chr1 
                 11 
                 204518240 
                 204527248 
                 204518561 
                 204519094 
                 534 
                 AAGCATGGGAGAACAGTTAG 
                  64 
                 AAATGTGCATGGAAGAAATC 
                 217 
                  56 
               
               
                   
               
               
                 MDM4 
                 chr1 
                 11 
                 204518240 
                 204527248 
                 204519011 
                 204519570 
                 560 
                 TACTTTATGCAGCAGTCAGG 
                  65 
                 CTATAATCCCAGCAATTTGG 
                 218 
                  57 
               
               
                   
               
               
                 MDM4 
                 chr1 
                 11 
                 204518240 
                 204527248 
                 204519551 
                 204520101 
                 551 
                 CCAAATTGCTGGGATTATAG 
                  66 
                 AAGACATGTTCTGACGGAAG 
                 219 
                  58 
               
               
                   
               
               
                 MDM4 
                 chr1 
                 11 
                 204518240 
                 204527248 
                 204519982 
                 204520495 
                 514 
                 CCCTGGGACTATAGATTTAGC 
                  67 
                 ATGACTCCTAAGACGCAAAG 
                 220 
                  59 
               
               
                   
               
               
                 MDM4 
                 chr1 
                 11 
                 204518240 
                 204527248 
                 204520474 
                 204521069 
                 596 
                 CTCTTTGCGTCTTAGGAGTC 
                  68 
                 GTGGTCCAAGACAATTCTTC 
                 221 
                  60 
               
               
                   
               
               
                 MDM4 
                 chr1 
                 11 
                 204518240 
                 204527248 
                 204520897 
                 204521454 
                 558 
                 TGCAGAGACTGATCTTTGAG 
                  69 
                 ACCAACAACGACATTATGAG 
                 222 
                  61 
               
               
                   
               
               
                 MDM4 
                 chr1 
                 11 
                 204518240 
                 204527248 
                 204521434 
                 204521966 
                 533 
                 TCTCATAATGTCGTTGTTGG 
                  70 
                 GTAAAGATGAAATTCGGCTC 
                 223 
                  62 
               
               
                   
               
               
                 MDM4 
                 chr1 
                 11 
                 204518240 
                 204527248 
                 204521808 
                 204522394 
                 587 
                 TTGATCCTAAATTTGACACATC 
                  71 
                 GCCTTGCTTTAGTTTAGTGG 
                 224 
                  63 
               
               
                   
               
               
                 MDM4 
                 chr1 
                 11 
                 204518240 
                 204527248 
                 204522261 
                 204522731 
                 471 
                 AAAGTGCTGAGATTACAGGC 
                  72 
                 TGGTAATGTGGTGTGATTTC 
                 225 
                  64 
               
               
                   
               
               
                 MDM4 
                 chr1 
                 11 
                 204518240 
                 204527248 
                 204522686 
                 204523254 
                 596 
                 GCAACGTGCTGTAGACTATG 
                  73 
                 ATTGCATTGAATTGACACAC 
                 226 
                  65 
               
               
                   
               
               
                 MDM4 
                 chr1 
                 11 
                 204518240 
                 204527248 
                 204523103 
                 204523650 
                 548 
                 CAAGCATTTGAAATATGCAG 
                  74 
                 TCACGTTTGGTACATGAGAC 
                 227 
                  66 
               
               
                   
               
               
                 MDM4 
                 chr1 
                 11 
                 204518240 
                 204527248 
                 204523496 
                 204524044 
                 549 
                 TTAGTTCTGATGGTTCTCCC 
                  75 
                 TGCTGTATTCACCAATAACG 
                 228 
                  67 
               
               
                   
               
               
                 MDM4 
                 chr1 
                 11 
                 204518240 
                 204527248 
                 204523931 
                 204524513 
                 583 
                 TATAGGAGCCATTGGATTTC 
                  76 
                 GTCAGGAGATCAAGACCATC 
                 229 
                  68 
               
               
                   
               
               
                 MDM4 
                 chr1 
                 11 
                 204518240 
                 204527248 
                 204524182 
                 204524677 
                 496 
                 ATCTGAAATCCAAGATGCTG 
                  77 
                 TACAGCAACTGCTCTGAAAG 
                 230 
                  69 
               
               
                   
               
               
                 MDM4 
                 chr1 
                 11 
                 204518240 
                 204527248 
                 204524537 
                 204525135 
                 599 
                 TCCCAAAGTACTGGGATTAC 
                  78 
                 ATTTGCTACTGTTGACAGGG 
                 231 
                  70 
               
               
                   
               
               
                 MDM4 
                 chr1 
                 11 
                 204518240 
                 204527248 
                 204525034 
                 204525491 
                 458 
                 ATTTCTTATCTGAAGGCACTG 
                  79 
                 CATCACACACAGAAAGGAAG 
                 232 
                  71 
               
               
                   
               
               
                 MDM4 
                 chr1 
                 11 
                 204518240 
                 204527248 
                 204525312 
                 204525853 
                 542 
                 TACCAAAGACCCTTATCAGC 
                  80 
                 TTCTGTAAGAAGGAAGCCTG 
                 233 
                  72 
               
               
                   
               
               
                 MDM4 
                 chr1 
                 11 
                 204518240 
                 204527248 
                 204525814 
                 204526369 
                 556 
                 TGTCTCAAAGAAATTGAGGTC 
                  81 
                 AGTAATCAAACAGGCTCTGC 
                 234 
                  73 
               
               
                   
               
               
                 MDM4 
                 chr1 
                 11 
                 204518240 
                 204527248 
                 204526066 
                 204526663 
                 598 
                 TAAGTGCCTCTTGGGTAGAG 
                  82 
                 AGCTACTTGAGAGGTTGAGG 
                 235 
                  74 
               
               
                   
               
               
                 MDM4 
                 chr1 
                 11 
                 204518240 
                 204527248 
                 204526557 
                 204527101 
                 545 
                 GTCTTACTCTGTCACCCAGG 
                  83 
                 CTTTCCTCATCTAGTGAGCTG 
                 236 
                  75 
               
               
                   
               
               
                 MDM4 
                 chr1 
                 11 
                 204518240 
                 204527248 
                 204526920 
                 204527482 
                 563 
                 TCAGAGAATCACAAGAGCAG 
                  84 
                 GATGGATTTCTTCAGGATTG 
                 237 
                  76 
               
               
                   
               
               
                 MYC 
                 chr8 
                  1 
                 128748314 
                 128748869 
                 128748285 
                 128748719 
                 435 
                 CTTTATAATGCGAGGGTCTG 
                  85 
                 TTGTAAGTTCCAGTGCAAAG 
                 238 
                  77 
               
               
                   
               
               
                 MYC 
                 chr8 
                  1 
                 128748314 
                 128748869 
                 128748485 
                 128748945 
                 461 
                 GTAGTAATTCCAGCGAGAGG 
                  86 
                 ATTTAGGCATTCGACTCATC 
                 239 
                  78 
               
               
                   
               
               
                 MYC 
                 chr8 
                  2 
                 128750493 
                 128751265 
                 128750452 
                 128750908 
                 457 
                 TTTAACTCAAGACTGCCTCC 
                  87 
                 TACAGTCCTGGATGATGATG 
                 240 
                  79 
               
               
                   
               
               
                 MYC 
                 chr8 
                  2 
                 128750493 
                 128751265 
                 128750834 
                 128751381 
                 548 
                 ACATGGTGAACCAGAGTTTC 
                  88 
                 TCCAGATCTGCTATCTCTCC 
                 241 
                  80 
               
               
                   
               
               
                 MYC 
                 chr8 
                  3 
                 128752641 
                 128753680 
                 128752528 
                 128752893 
                 366 
                 GTCCAGAGACCTTTCTAACG 
                  89 
                 TGATCTGTCTCAGGACTCTG 
                 242 
                  88 
               
               
                   
               
               
                 MYC 
                 chr8 
                  3 
                 128752641 
                 128753680 
                 128752715 
                 128753285 
                 571 
                 AGAGTCTGGATCACCTTCTG 
                  90 
                 TTTGATCATGCATTTGAAAC 
                 243 
                  86 
               
               
                   
               
               
                 MYC 
                 chr8 
                  3 
                 128752641 
                 128753680 
                 128753173 
                 128753687 
                 515 
                 AACTTGAACAGCTACGGAAC 
                  91 
                 TCACAACTTAAGATTTGGCTC 
                 244 
                  87 
               
               
                   
               
               
                 MYCL 
                 chr1 
                  1 
                  40367479 
                  40367687 
                  40367327 
                  40367715 
                 389 
                 AGCGAGTTCAAAGCAAACTT 
                  92 
                 GCGACGAGATATAAGGCAGT 
                 245 
                  81 
               
               
                   
               
               
                 MYCL 
                 chr1 
                  2 
                  40366610 
                  40367115 
                  40366514 
                  40367080 
                 567 
                 AGAGCTTGAGAAGAGCCAAT 
                  93 
                 TTTCTACGACTATGACTGCG 
                 246 
                  82 
               
               
                   
               
               
                 MYCL 
                 chr1 
                  2 
                  40366610 
                  40367115 
                  40367010 
                  40367346 
                 337 
                 ATTTCTTCCAGATGTCCTCG 
                  94 
                 AAGTTTGCTTTGAACTCGCT 
                 247 
                  83 
               
               
                   
               
               
                 MYCL 
                 chr1 
                  3 
                  40361095 
                  40363642 
                  40360973 
                  40361525 
                 553 
                 GAGTGGAATGACCAGGTTAG 
                  95 
                 ATGGTTTCTTTCTGAGGTTG 
                 248 
                  84 
               
               
                   
               
               
                 MYCL 
                 chr1 
                  3 
                  40361095 
                  40363642 
                  40361453 
                  40362039 
                 587 
                 AGGGTAGAGAGGCTATTTCC 
                  96 
                 TTTGAAGTTCTTCTGGAACC 
                 249 
                  85 
               
               
                   
               
               
                 MYCL 
                 chr1 
                  3 
                  40361095 
                  40363642 
                  40362026 
                  40362521 
                 496 
                 AGAAGAACTTCAAACTTGCC 
                  97 
                 CATTGACCATTACCTCACTG 
                 250 
                  89 
               
               
                   
               
               
                 MYCL 
                 chr1 
                  3 
                  40361095 
                  40363642 
                  40362463 
                  40362896 
                 434 
                 TAAAGGTTTCCAACTCCTTG 
                  98 
                 AATAAAGGCTTGCATTCTTG 
                 251 
                  90 
               
               
                   
               
               
                 MYCL 
                 chr1 
                  3 
                  40361095 
                  40363642 
                  40363271 
                  40363855 
                 585 
                 CCAGGAAGTTGTGATTCTTC 
                  99 
                 TTTCCTTCTTGCTAATGTCC 
                 252 
                  91 
               
               
                   
               
               
                 MYCN 
                 chr2 
                  1 
                  16080559 
                  16081175 
                  16080527 
                  16081017 
                 491 
                 TTTTTATGGAAATCAGGAGG 
                 100 
                 ACCCAGAGATGGTTTTGTTT 
                 253 
                  92 
               
               
                   
               
               
                 MYCN 
                 chr2 
                  1 
                  16080559 
                  16081175 
                  16080642 
                  16081165 
                 524 
                 GTTAATAATATCCCCCGAGC 
                 101 
                 ACAGCTCAAACACAGACAGA 
                 254 
                  93 
               
               
                   
               
               
                 MYCN 
                 chr2 
                  1 
                  16080559 
                  16081175 
                  16081147 
                  16081538 
                 392 
                 CTGTCTGTGTTTGAGCTGTC 
                 102 
                 AACAACAGACACCCATATCC 
                 255 
                  94 
               
               
                   
               
               
                 MYCN 
                 chr2 
                  2 
                  16082069 
                  16082976 
                  16081882 
                  16082346 
                 465 
                 AGCTTGTACACAAAAGGAGG 
                 103 
                 CAAACTTCTTCCAGATGTCC 
                 256 
                  95 
               
               
                   
               
               
                 MYCN 
                 chr2 
                  2 
                  16082069 
                  16082976 
                  16082241 
                  16082780 
                 540 
                 CTCGAGTTTGACTCGCTACA 
                 104 
                 GTTCACGGGAAAGGGGAAGA 
                 257 
                  96 
               
               
                   
               
               
                 MYCN 
                 chr2 
                  2 
                  16082069 
                  16082976 
                  16082425 
                  16082985 
                 561 
                 AGATGCTGCTTGAGAACGAG 
                 105 
                 GGTCTTTACCTGAATCGCTC 
                 258 
                  97 
               
               
                   
               
               
                 MYCN 
                 chr2 
                  3 
                  16058614 
                  16087129 
                  16085471 
                  16086069 
                 599 
                 ACATCTATGTTGATGGACCC 
                 106 
                 CTCATTCTTTACCAACTCCG 
                 259 
                  98 
               
               
                   
               
               
                 MYCN 
                 chr2 
                  3 
                  16085614 
                  16087129 
                  16086055 
                  16086635 
                 581 
                 TTGGTAAAGAATGAGAAGGC 
                 107 
                 TGTCAATGGTATTTACAGAAATG 
                 260 
                  99 
               
               
                   
               
               
                 MYCN 
                 chr2 
                  3 
                  16085614 
                  16087129 
                  16086508 
                  16087031 
                 524 
                 GTTCCAAGTTTCCAAACAAC 
                 108 
                 AGAACTTTGCATTTACCCAG 
                 261 
                 100 
               
               
                   
               
               
                 MYCN 
                 chr2 
                  3 
                  16085614 
                  16087129 
                  16087008 
                  16087449 
                 442 
                 AGAACTGGGTAAATGCAAAG 
                 109 
                 TGAGGTCTCAGCTTAATTCC 
                 262 
                 101 
               
               
                   
               
               
                 NCOA3 
                 chr20 
                  1 
                  46130600 
                  46130763 
                  46130398 
                  46130992 
                 595 
                 AAAAATTAAGGGCAGGGCTA 
                 110 
                 AGCTTCGTCTCAGCTCCTAC 
                 263 
                 102 
               
               
                   
               
               
                 NCOA3 
                 chr20 
                  2 
                  46211926 
                  46212005 
                  46211894 
                  46212483 
                 590 
                 AAATTCAATCCCTCCTCTTC 
                 111 
                 AGGTGATCTAACCACCTCAG 
                 264 
                 103 
               
               
                   
               
               
                 NCOA3 
                 chr20 
                  3 
                  46250972 
                  46251074 
                  46250747 
                  46251198 
                 452 
                 GGAACATTTCTGTCTTGGAG 
                 112 
                 ACTTACCACGAAGTGAAACC 
                 265 
                 104 
               
               
                   
               
               
                 NCOA3 
                 chr20 
                  4 
                  46252654 
                  46252827 
                  46252552 
                  46253120 
                 569 
                 GTAATCATGTAATAGTGTTG 
                 113 
                 GATCTGTCACAGTTTCTCCC 
                 266 
                 105 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 TATAGGG 
                   
                   
                   
                   
               
               
                   
               
               
                 NCOA3 
                 chr20 
                  5 
                  46254124 
                  46254225 
                  46253918 
                  46254512 
                 595 
                 TTAGGTATCTTCTGGCTTCC 
                 114 
                 TACAGGCTACCTTTCCTTTC 
                 267 
                 106 
               
               
                   
               
               
                 NCOA3 
                 chr20 
                  6 
                  46255745 
                  46255920 
                  46255621 
                  46256183 
                 563 
                 TTACCTCCTTGAAGGTCTTG 
                 115 
                 ATTTCAGGCTGGCAATATAC 
                 268 
                 107 
               
               
                   
               
               
                 NCOA3 
                 chr20 
                  7 
                  46256304 
                  46256493 
                  46256232 
                  46256752 
                 521 
                 CTTGAATTCTTGATGATGGTC 
                 116 
                 TGGTAATAAAGCTCTCAGGG 
                 269 
                 108 
               
               
                   
               
               
                 NCOA3 
                 chr20 
                  8 
                  46256665 
                  46256767 
                  46256391 
                  46256976 
                 586 
                 ATTCTGGAAGACATAAACGC 
                 117 
                 AACATACCCAATTCAAATGC 
                 270 
                 109 
               
               
                   
               
               
                 NCOA3 
                 chr20 
                  9 
                  46262239 
                  46262380 
                  46262160 
                  46262475 
                 316 
                 CAGTGCTAAGCCATGTGTAG 
                 118 
                 TAAATCCAGGAGTTCGAGTC 
                 271 
                 110 
               
               
                   
               
               
                 NCOA3 
                 chr20 
                 10 
                  46262791 
                  46262939 
                  46262711 
                  46263063 
                 353 
                 GTATATTTCCTCCCTGTCCC 
                 119 
                 CATCAAACCCAATAACCTTC 
                 272 
                 111 
               
               
                   
               
               
                 NCOA3 
                 chr20 
                 11 
                  46264065 
                  46264457 
                  46263932 
                  46264470 
                 539 
                 CAAAGTGCTGGGAATATAGG 
                 120 
                 TCAACACAAATACCTGCAAC 
                 273 
                 112 
               
               
                   
               
               
                 NCOA3 
                 chr20 
                 12 
                  46264634 
                  46265506 
                  46264235 
                  46264834 
                 600 
                 ATGAGTGGAGCTAGGTATGG 
                 121 
                 CTTGGAATCCTGATTGCTTA 
                 274 
                 113 
               
               
                   
               
               
                 NCOA3 
                 chr20 
                 12 
                  46264634 
                  46265506 
                  46264800 
                  46265238 
                 439 
                 CCCAACCAAGTAAAGTAAGC 
                 122 
                 GCAGTAATCTTGGCTACCTC 
                 275 
                 114 
               
               
                   
               
               
                 NCOA3 
                 chr20 
                 12 
                  46264634 
                  46265506 
                  46265206 
                  46265783 
                 578 
                 GAATTCACCAGCTGAGGTAG 
                 123 
                 CTCTTAATGACCCAATCTGC 
                 276 
                 115 
               
               
                   
               
               
                 NCOA3 
                 chr20 
                 13 
                  46266391 
                  46266527 
                  46266333 
                  46266855 
                 523 
                 TGTTTATACCTGTGTGTCTGG 
                 124 
                 TTAATCCAGTTCTCTGTGGC 
                 277 
                 116 
               
               
                   
               
               
                 NCOA3 
                 chr20 
                 14 
                  46267751 
                  46267946 
                  46267493 
                  46268028 
                 536 
                 AGTTCTCAGTACTTCAGCCG 
                 125 
                 CTCCCAATTATTTAGATGGC 
                 278 
                 117 
               
               
                   
               
               
                 NCOA3 
                 chr20 
                 15 
                  46268320 
                  46268566 
                  46268163 
                  46268576 
                 414 
                 ATAGTGGCCTATGTCTCCAC 
                 126 
                 GGACACTTACTCATTTGAAGC 
                 279 
                 118 
               
               
                   
               
               
                 NCOA3 
                 chr20 
                 16 
                  46268668 
                  46268795 
                  46268503 
                  46268943 
                 441 
                 CGGTCTAATAGCATACCAGG 
                 127 
                 AGAGTTACACGAGAAATGCC 
                 280 
                 119 
               
               
                   
               
               
                 NCOA3 
                 chr20 
                 17 
                  46270956 
                  46271128 
                  46270628 
                  46271179 
                 552 
                 AGGAGTATCTTCTCCCATCC 
                 128 
                 GCGCACACACACAAATATAC 
                 281 
                 120 
               
               
                   
               
               
                 NCOA3 
                 chr20 
                 18 
                  46275816 
                  46276110 
                  46275557 
                  46276087 
                 531 
                 CACAGTACACCTGGTTCTTG 
                 129 
                 GAAGCTGCATTCTAAGTTGC 
                 282 
                 121 
               
               
                   
               
               
                 NCOA3 
                 chr20 
                 18 
                  46275816 
                  46276110 
                  46275868 
                  46276458 
                 591 
                 GTAATGATGGATCAGAAGGC 
                 130 
                 AAATGCTGAAATCAAGAAGG 
                 283 
                 122 
               
               
                   
               
               
                 NCOA3 
                 chr20 
                 19 
                  46277748 
                  46277853 
                  46277654 
                  46278204 
                 551 
                 GATATTACCTCATTGGCTGG 
                 131 
                 TGCATGTTGTTTCATAATCC 
                 284 
                 123 
               
               
                   
               
               
                 NCOA3 
                 chr20 
                 20 
                  46279728 
                  46280020 
                  46279700 
                  46280285 
                 586 
                 TAATTGCACTCTTTCTTGGG 
                 132 
                 AACTTTGCAGTGTTTCTTCC 
                 285 
                 124 
               
               
                   
               
               
                 NCOA3 
                 chr20 
                 21 
                  46281149 
                  46281324 
                  46281096 
                  46281383 
                 288 
                 TTCTAAGGAGAAGGCATTTG 
                 133 
                 TAAGTTCTTGGACTTCTGGG 
                 286 
                 125 
               
               
                   
               
               
                 NCOA3 
                 chr20 
                 22 
                  46281674 
                  46281816 
                  46281629 
                  46282021 
                 393 
                 GCTAAAGTGACTTCCAGAGG 
                 134 
                 GAGATCCCATCTTACAATGC 
                 287 
                 126 
               
               
                   
               
               
                 NCOA3 
                 chr20 
                 23 
                  46282149 
                  46285621 
                  46282008 
                  46282592 
                 585 
                 TAAGATGGGATCTCAGGAAC 
                 135 
                 TCTTTGTCCAATACTGCAAC 
                 288 
                 127 
               
               
                   
               
               
                 NCOA3 
                 chr20 
                 23 
                  46282149 
                  46285621 
                  46282430 
                  46282949 
                 520 
                 ATTCTGGAGACATGGAGTGT 
                 136 
                 AACCAGGAATGTGTTTCACT 
                 289 
                 128 
               
               
                   
               
               
                 NCOA3 
                 chr20 
                 23 
                  46282149 
                  46285621 
                  46282912 
                  46283260 
                 349 
                 TTGAGGTCTTGAGGGAATAG 
                 137 
                 ACCACACAGCTTACTGAAATC 
                 290 
                 129 
               
               
                   
               
               
                 NCOA3 
                 chr20 
                 23 
                  46282149 
                  46285621 
                  46283242 
                  46283793 
                 552 
                 TTTCAGTAAGCTGTGTGGTG 
                 138 
                 AGGGACATAATGAAAGCATC 
                 291 
                 230 
               
               
                   
               
               
                 NCOA3 
                 chr20 
                 23 
                  46282149 
                  46285621 
                  46283688 
                  46284229 
                 542 
                 GACCTGAATCCCATATTGAG 
                 139 
                 GTGGGTCTGGAAATAATCAG 
                 292 
                 131 
               
               
                   
               
               
                 NCOA3 
                 chr20 
                 23 
                  46282149 
                  46285621 
                  46284210 
                  46284671 
                 462 
                 CTGATTATTTCCAGACCCAC 
                 140 
                 AGAAATCTTGAGTTTGCACC 
                 293 
                 139 
               
               
                   
               
               
                 NCOA3 
                 chr20 
                 23 
                  46282149 
                  46285621 
                  46284324 
                  46284768 
                 445 
                 AAATCCGAAAACTTCCATTG 
                 141 
                 GAGGAGAGGTAGACAGCAGG 
                 294 
                 137 
               
               
                   
               
               
                 NCOA3 
                 chr20 
                 23 
                  46282149 
                  46285621 
                  46284746 
                  46285291 
                 546 
                 ACTCCTGCTGTCTACCTCTC 
                 142 
                 TGCTCCTAGGAACCTAATTG 
                 295 
                 138 
               
               
                   
               
               
                 NCOA3 
                 chr20 
                 23 
                  46282149 
                  46285621 
                  46285161 
                  46285693 
                 533 
                 AGTTCTTTGATCCAGAGGTG 
                 143 
                 TTCCTTAACCTCCTTTACCC 
                 296 
                 132 
               
               
                   
               
               
                 NKX2-1 
                 chr14 
                  1 
                  36989257 
                  36989430 
                  36989105 
                  36989609 
                 505 
                 AGGAGAGATGGTTGAGAGGA 
                 144 
                 ACTGAAAAACCCCTGAGCTG 
                 297 
                 133 
               
               
                   
               
               
                 NKX2-1 
                 chr14 
                  2 
                  36988189 
                  36988575 
                  36987990 
                  36988496 
                 507 
                 GCTACCAAGTGCCTGTTCTT 
                 145 
                 AGCTACAAGAAAGTGGGCAT 
                 298 
                 134 
               
               
                   
               
               
                 NKX2-1 
                 chr14 
                  2 
                  36988189 
                  36988575 
                  36988249 
                  36988667 
                 422 
                 TTCCTCATGGTGTCCTGGTA 
                 146 
                 ACCAGAATATTTGGCAAAGG 
                 299 
                 135 
               
               
                   
               
               
                 NKX2-1 
                 chr14 
                  3 
                  36985603 
                  36987225 
                  36985377 
                  36985969 
                 593 
                 ACTGCTCAAGATTTGTTTCC 
                 147 
                 TCACTGACACAAAGGAAGTG 
                 300 
                 136 
               
               
                   
               
               
                 NKX2-1 
                 chr14 
                  3 
                  36985603 
                  36987225 
                  36985737 
                  36986227 
                 491 
                 TACACAGATTTGTCAATGCC 
                 148 
                 ATCTTTAAGCAGAGAAGGGC 
                 301 
                 140 
               
               
                   
               
               
                 NKX2-1 
                 chr14 
                  3 
                  36985603 
                  36987225 
                  36986160 
                  36986513 
                 354 
                 GAAAACCCATTTGAATCACC 
                 149 
                 CTCCACCTTGCTATACGGTC 
                 302 
                 141 
               
               
                   
               
               
                 NKX2-1 
                 chr14 
                  3 
                  36985603 
                  36987225 
                  36986374 
                  36986970 
                 597 
                 TGTTAAGAAAAGTCGAAGCG 
                 150 
                 AGAACCACCGCTACAAAATG 
                 303 
                 142 
               
               
                   
               
               
                 NKX2-1 
                 chr14 
                  3 
                  36985603 
                  36987225 
                  36986967 
                  36987556 
                 590 
                 TTCTGGAACCAGATCTTGAC 
                 151 
                 TAATCCTAATGCTCTGACCC 
                 304 
                 143 
               
               
                   
               
               
                 SKP2 
                 chr5 
                  1 
                  36152144 
                  36152372 
                  36152137 
                  36152620 
                 484 
                 GAAACTACAATTCCCAGCAG 
                 152 
                 GAGAGACAGGGCAATCATAC 
                 305 
                 144 
               
               
                   
               
               
                 SKP2 
                 chr5 
                  2 
                  36152872 
                  36153144 
                  36152615 
                  36153148 
                 534 
                 TCTCTCTCCTTGTCTGTTCC 
                 153 
                 TTACCTGGAAAGTTCTCTCG 
                 306 
                 145 
               
               
                   
               
               
                 SKP2 
                 chr5 
                  3 
                  36163746 
                  36163858 
                  36163699 
                  36164087 
                 389 
                 GATAGGGTGAAAGAATGGTG 
                 154 
                 ACTGAATACAGGGCAAAGAG 
                 307 
                 146 
               
               
                   
               
               
                 SKP2 
                 chr5 
                  4 
                  36166620 
                  36166764 
                  36166512 
                  36167017 
                 504 
                 GCTTCAAGGAGATTTAGCAG 
                 155 
                 AAGACAAATGTGCCTCTTTC 
                 308 
                 147 
               
               
                   
               
               
                 SKP2 
                 chr5 
                  5 
                  36168414 
                  36168549 
                  36168352 
                  36168852 
                 501 
                 GTTTGAAATTGGATGTACCC 
                 156 
                 CAGCATTCACTAACAAGGTG 
                 309 
                 148 
               
               
                   
               
               
                 SKP2 
                 chr5 
                  6 
                  36170445 
                  36170544 
                  36170281 
                  36170703 
                 423 
                 GAGGCAAATTATCCTGTTTG 
                 157 
                 TTGGACAGAAAGTTAGGAGG 
                 310 
                 149 
               
               
                   
               
               
                 SKP2 
                 chr5 
                  7 
                  36171704 
                  36171835 
                  36171414 
                  36171948 
                 535 
                 AAGACTGGCATTTCTACCTG 
                 158 
                 CATGCACTGGATTAAATGAG 
                 311 
                 150 
               
               
                   
               
               
                 SKP2 
                 chr5 
                  8 
                  36177066 
                  36177118 
                  36176945 
                  36177324 
                 380 
                 GTGTGGTTCTAATTGCATTG 
                 159 
                 ATTCCTGAAAGCAGTCATTC 
                 312 
                 151 
               
               
                   
               
               
                 SKP2 
                 chr5 
                  9 
                  36177286 
                  36177394 
                  36177180 
                  36177543 
                 364 
                 GGGAAAGGATCATAATGTTG 
                 160 
                 CTCTGCTGGTCTTTCATAGC 
                 313 
                 152 
               
               
                   
               
               
                 SKP2 
                 chr5 
                 10 
                  36183941 
                  36184142 
                  36183823 
                  36184304 
                 482 
                 TGCCTTTATCTGCTTAGACC 
                 161 
                 CAAGCATATGAAGTAGATGGG 
                 314 
                 153 
               
               
                   
               
            
           
         
       
     
     In some cases, amplification primers designed to amplify a portion of a human genome targeted by one or more of the FISH probes (e.g., a FISH probe set forth in Table 3) can be used in a single assay as described herein. For example, amplification primers designed to amplify a portion of a human genome targeted by 5, 10, 20, or more FISH probes can be used in a single assay as described herein. In some cases, two or more different amplification primer pairs can be designed to amplify different portions of the same region of a human genome targeted by one of a FISH probe. For example, three primer pairs can be designed to amplify three different regions of the first FISH probe listed in Table 3. In some cases, as described herein, nucleic acid capture techniques can be used in addition to or in place of amplification techniques to increase sequence read coverage. 
     The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims. 
     EXAMPLES 
     Example 1—Combining LC-WGS and Targeted Nucleic Acid Amplification to Improve the Interpretation of Cancer Panel Tests 
     The combination of LC-WGS and targeted nucleic acid amplification is used to improve the clinical interpretation of Cancer Panel Tests that focus primarily on identifying mutations driving tumorgenesis in targeted regions of the genome. LC-WGS provides information in the genome wide nature and location of amplifications and deletions. This information is used to assess the aggressiveness of the tumor and/or provide additional support to the mutations reported in the targeted regions. 
     The values of combining LC-WGS and targeted nucleic acid amplification was highlighted by performing the following. Whole DNA of biospecimens was extracted. Targeted regions amplification was performed using an amplicon-based protocol to allow variant calling. The targeted amplification was performed using a number of cycles that was protocol specific, but might vary from protocol to protocol (15 to 20 cycles). The amplification was done below saturation level, leaving in solution about 25% of reads that do not map to the target regions but map the remaining areas of the genome. Upon sequencing, 3.5M reads in total were obtained. The 2.6M reads mapping the target regions were extracted and processed for variant calling using a DNA processing workflow. The high coverage of these regions (at about 1000× average coverage) allowed for clinical grade variants calling. For the two ovarian samples displayed on  FIGS. 4A and 4B , mutations in the DNA repair and signal transduction genes were reported in the clinical report. 
     The 0.9M remaining reads (not mapped to these targeted regions) were processed. The resulting aligned reads were clustered in 10 kb bins. The count of the reads in each bin was displayed on  FIGS. 4A and 4B  for two ovarian tumor samples. The plots clearly highlighted chromosome level amplifications and target local amplifications that can be used to further refine the interpretation of the mutations. For example, the balance of chromosome or chromosome arm amplification and local amplification can be informative of the aggressiveness of the tumor. 
     Example 2—Combining LC-WGS and Targeted Nucleic Acid Amplification to Replace a FISH Assay 
     FISH assays are commonly used by clinical laboratories to report the presence of cancer cells in cytology specimens. For example, the UroVysion FISH assay (Abbott Molecular Inc.) is used to identify cancer cells in urine and biliary samples. This FISH assay includes a set of four fluorescent probes that target the chromosomal location 9p21 and the centromeres of chromosomes 3, 7, and 17. Probes targeting chromosomal locations are used to report amplifications and deletions in these regions. The ones targeting centromeres identify the loss or the presence of additional copies of chromosomes. 
     For lung and pleural samples, the LaVysion FISH assay (Abbott Molecular Inc.) is used. The four fluorescent probes of this assay target chromosomal locations 7p12, 5p12, 8q24, and the centromere of chromosome 6. Each of these FISH probes is greater than 150,000 bases. The probes are of large size to ensure that their luminescence is high enough to be observed under a microscope. 
     An assay is designed as described herein to identify deletions and/or amplifications in the genomic regions targeted by the FISH probes, while also having the ability to provide a low resolution global view of alterations across the genome. The regions amplified by these primers overlap with the ones targeted by the FISH probes. The amplified regions are not the same size as the FISH probes since the FISH probes are often greater than 150,000 bases long for technical reasons that are specific to the FISH assay. The FISH probes that target centromeres identify whole chromosome amplifications and/or deletions, which will be identified by the LC-WGS of the designed assay. 
     In particular, the designed assay combines both the UroVysion and LaVysion in a single assay as set forth in Table 3. Table 3 provides a list of primers that are used to amplify genomic regions 9p21, 7p12, 5p12, and 8q24. The design of these primers was optimized for a melting temperature of about 60 degrees. Primers for the FISH probes targeting centromere regions were not included since the LC-WGS component of the designed assay can identify genomic amplifications and/or deletions of whole chromosomes. Table 3 provides in the 1st column the cytoband location of the regions amplified by the primers followed by the genomic start and end coordinates of the region amplified by the primers, the length of the amplified genomic region, and the sequence of the forward and reverse primers. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Example of the design of 
               
               
                 an assay that replaces two FISH assays. 
               
            
           
           
               
               
               
               
               
               
            
               
                 Cytoband 
                 start 
                 end 
                 length 
                 forward 
                 reverse 
               
               
                   
               
               
                 9p21 
                  26549942 
                  26550536 
                 595 
                 GTCTGGTTCTGGCT 
                 GCCACCTCCTCTTT 
               
               
                   
                   
                   
                   
                 CTGTGC 
                 GTCAGC 
               
               
                   
                   
                   
                   
                 (SEQ ID NO: 1) 
                 (SEQ ID NO: 2) 
               
               
                   
               
               
                 7p12 
                  51867623 
                  51868189 
                 567 
                 AAGAGTTGCCAAGG 
                 TGACAGGCTTGAAT 
               
               
                   
                   
                   
                   
                 CACGAC 
                 GCACCC 
               
               
                   
                   
                   
                   
                 (SEQ ID NO: 3) 
                 (SEQ ID NO: 4) 
               
               
                   
               
               
                 5p12 
                  43864904 
                  43865490 
                 587 
                 AGACTTCACCTTTG 
                 CCTGGAGAACAGGA 
               
               
                   
                   
                   
                   
                 GTGCCC 
                 TGCGAC 
               
               
                   
                   
                   
                   
                 (SEQ ID NO: 5) 
                 (SEQ ID NO: 6) 
               
               
                   
               
               
                 8q24 
                 130915820 
                 130916382 
                 563 
                 TTCAACCAACCCAT 
                 TTCATGGCCACCAC 
               
               
                   
                   
                   
                   
                 CAGCGG 
                 AATGGC 
               
               
                   
                   
                   
                   
                 (SEQ ID NO: 7) 
                 (SEQ ID NO: 8) 
               
               
                   
               
            
           
         
       
     
     Example 3—Single Assay for the Combined Reporting for Fetal Fraction Estimation and the Presence to Fetal Trisomy from the Blood of the Mother 
     LC-WGS sequencing has been successfully applied to the detection fetal trisomy from the blood of pregnant women. However, to optimize the selectivity and sensitivity of LC-WGS, an additional test is needed to measure the fetal fraction. This additional test can be implemented using SNP microarrays to measure the allelic imbalance. Some in silico approaches (e.g., bioinformatics) also have been used for the same purpose. 
     An assay is designed as described herein to identify in a single assay both the fetal fraction in the blood of the mother and the presence of fetal trisomy. For this assay, the amplified regions are designed to target SNPs empirically selected to maximize the likelihood to be heterogeneous in the fetus and homozygous in the mother. The ratio of the reads mapped to the major and minor allele is informative of the fraction of the DNA from the fetus present in the blood of the mother. Calling the genotypes of SNPs is not possible from LC-WGS alone since this technique does not have enough reads available to call genotypes. 
     Example 4—Combining LC-WGS and Targeted Nucleic Acid Amplification for the Early Detection of Cancer 
     The methods and materials provided herein are used for the early detection of cancer in cell free DNA. As tumors develop, a significant percentage of tumor cells die, shedding their abnormal DNA in the blood stream. The methods and materials provided herein are used to detect genomic amplification and/or deletion events in cell free DNA, thereby detecting the presence of a tumor. The low coverage whole genome sequencing of the assay provides a low resolution whole genome view of amplifications and/or deletions, while oncogenes frequently observed as being amplified and/or deleted across cancers are assessed at a higher sensitivity level using PCR amplification targeted regions. The following genes, which are frequently amplified across tumor types, are enriched as described herein: CCND1, LMO1, MDM2, MDM4, MYC, MYCL1, MYCN, NCOA3, NKX2-1, and SKP2. With the designed assay, multiple amplicons (e.g., about 5) of about 150 bp in length are assessed for each gene (for a total of about 50 amplicons per assay). Assuming that 400,000 reads of 150 bp is sequenced per sample, if 50 amplicons of 150 bp are used to amplify 50 regions of the genome, then each region exhibits a coverage of about 600× while the LC-WGS maintains an average coverage of about 1× for the DNA not enriched (Table 4). 
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 4 
               
               
                   
               
               
                   
                 read 
                 number of 
                 length of 
                 coverage of 
                   
               
               
                 reads per 
                 length 
                 amplified 
                 amplified 
                 the amplified 
                 LC-WGS 
               
               
                 sample 
                 (bp) 
                 regions 
                 regions 
                 regions 
                 coverage 
               
               
                   
               
             
            
               
                 30,000,000 
                 150 
                 50 
                 150 
                 600x 
                 1.0 
               
               
                   
               
            
           
         
       
     
     Other Embodiments 
     It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.