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- W2811157103 abstract "Next Generation Sequencing is increasingly used in translational cancer research and as a diagnostic test. Most tumor specimens available for testing are formalin-fixed, paraffin-embedded (FFPE) blocks. FFPE-derived DNA is typically damaged, causing difficulties for standard library preparation methods. Frayed ends prevent blunt-ended double-stranded adapter ligation, used for whole genome sequencing (WGS) and targeted bait hybridization library preparation. Fragmented DNA and modified bases interfere with PCR, which underlies amplification based targeted sequencing. We present a single-stranded library preparation method especially forgiving for low quality FFPE-derived DNA. By excising damaged bases and using single-stranded adapter ligation, we omit repair aimed at generating blunt-ended double-stranded DNA, and eliminate the need for whole genome PCR. After ligation of the first adapter, libraries can either undergo second adapter ligation to generate WGS libraries, or be used as input to oligo-selective sequencing (OS-Seq) target capture. We show that our FFPE-derived PCR-free WGS libraries exhibit coverage depth and uniformity highly similar to double-stranded PCR-free libraries of matched cell line (non-FFPE) materials. Additionally, we present performance of our WGS library preparation on clinical FFEP samples. We further demonstrate reproducible high and uniform coverage for reference standards and clinical FFPE samples in targeted capture. We show a lack of correlation between DNA quality and sequencing metrics such as on-target rate (R 2 =0.005), uniformity (R 2 =0.0002), and coverage (R 2 =0.06), with successful removal of C>T FFPE artifacts comparable to standard repair methods. We demonstrate sensitive copy number alteration detection through analysis of coverage depth, feasible due to the absence of pre-capture PCR. In conclusion, our single-stranded library preparation method is forgiving for sample quality and provides uniform and high coverage as the basis for somatic variant calling. Citation Format: Anna Vilborg. Single-stranded library preparation enables unbiased sequencing of damaged, FFPE-derived DNA for both targeted gene capture and WGS applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 422." @default.
- W2811157103 created "2018-07-10" @default.
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- W2811157103 date "2018-07-01" @default.
- W2811157103 modified "2023-10-14" @default.
- W2811157103 title "Abstract 422: Single-stranded library preparation enables unbiased sequencing of damaged, FFPE-derived DNA for both targeted gene capture and WGS applications" @default.
- W2811157103 doi "https://doi.org/10.1158/1538-7445.am2018-422" @default.
- W2811157103 hasPublicationYear "2018" @default.
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