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Comparison of custom capture for targeted next-generation DNA sequencing #
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Targeted, capture-based DNA sequencing은 유전체의 특정 영역만 시퀀싱함으로써 비용을 절감한다. NGS를 위한 4가지 상용 targeted sequencing 방법들을 비교한다.

4가지 방법은

  1. SureSelect
  2. HaloPlex
  3. Nextera
  4. SeqCap

비교 대상은

  1. uniformity
  2. ability to detect SNV and CNV

DNA fragmentation을 위해 sonication하는 것은 uniformity가 좋지만 다른 방법들은 준비시간이 짧은대신 uniformity가 나쁘다.

Summary #

Introduction #

경제성, deeper sequencing을 위해 custom capture gene panel이 필요함.

이상적인 방법은 높은 타게팅, uniformity가 필요함.

Materials and Methods #

Cell Lines and Tissue DNA Samples #

SureSelect Custome Target Enrichment Library Preparation #

HaloPlex Custom Exome Library Preparation #

Nextera Custom Enrichment Library Preparation #

SeqCap EZ Choice Library Preparation #

Sequencing of Libraries #

Illumina's MiSeq

SNP Array #

Genome-Wide Human SNP Array 6.0 for CNV

Mycoplasma Testing and Authentication #

Alignment #

  1. Adapter removed by MiSeq Reporter (SureSelect, Nextera, SeqCap) and SureCall (HaloPlex)
  2. Aligned by BWA
  3. BAM made by SAMtools
  4. Removed duplicate reads by MarkDuplicates of Picard
  5. Realigned around known indels in dbSNP by GATK
  6. Alignment statistics by bedtools

SNV Calling #

  1. SAMtools converts BAM to mpileup
  2. VarScan2 somitic function to VCF
  3. tumor purity using PurityEst -- reculate SNV
  4. Also calling by MuTect
  5. SNV annotation by ANNOVAR
  6. Compare SNV calling concordances (intersection / union)

CNV Calling #

  1. SAMtools converts BAM to mpileup
  2. VarScan2's copynumber function -> log2 ratio
  3. Genome-Wide Human SNP Array 6.0 calls -> log2 ratio
  4. Comparison -> get absolute CNV
  5. Correlation by technologies with SNP array of HCC-2218

Statistical Tests #

two-sided U-test

Collaboration with GenomOncology #

To ensure the reproducibility

Sequencing statistics were calculated

  • Inserted size by Picard CollectInsertSizeMetrics
  • statistics by Picard CollectAlignmentSummaryMetrics
  • complexity by Picard CollectAlignmentSummary
  • raw sequencing depth by GATK DepthOfCoverage

Results #

Library Construction and Probe Design Vary between Targeted DNA Capture Methods #

Comparison of Libraries and On-Target Sequencing Metrics #

Depth and Uniformity of On-Target Sequencing #

Impact of GC Content on Target Capture #

Using SNV Variant Fractions to Calculate Tumor Purity #

Comparison of CNV Calling with SNP Array #

Discussion #

Suggested Pages #