In:
Cancer Research, American Association for Cancer Research (AACR), Vol. 77, No. 13_Supplement ( 2017-07-01), p. 4019-4019
Abstract:
Next Generation Sequencing is increasingly implemented as a diagnostic test to identify actionable mutations in cancer patient samples. However, for routine diagnostics, tumor DNA is extracted from formalin-fixed, paraffin-embedded (FFPE) samples, which yields low quantity of damaged DNA. Inability to accurately repair the ends of these DNA fragments impairs adapter ligation by standard double stranded ligation methods. The resulting low yield of adapter-ligated DNA introduces the need for whole-genome PCR amplification prior to target capture. The drawback of such PCR amplification is the introduction of PCR biases, causing reduced sensitivity in the detection of copy number alterations (CNAs), an important biomarker for targeted therapy. To address the need for a library preparation platform that performs well with low quality and quantity DNA, and without relying on massive PCR amplification, we developed an improved, in-solution, version the OS-Seq targeted enrichment assay. OS-Seq circumvents the reliance on PCR amplification by using a single-stranded adapter ligation approach. Damaged bases induced by formalin fixation are removed by excision instead of attempting repair, and then DNA is denatured prior to adapter ligation. This method of adapter ligation result in yields of ~50% for low quality samples, eliminating the need for whole genome PCR. OS-Seq directly uses the adapter-ligated DNA in a linear targeted primer-extension, followed by low-cycle post-capture PCR expansion with Illumina bridge-PCR primers prior to library sequencing. We investigated the PCR duplication rate of the OS-Seq libraries by including an 11-mer random barcode to track unique molecules. We found that most input molecules were present in the sequencing reads at only one copy. Further, we demonstrate a linear correlation between the amount of DNA input (ranging from 1 to 600 ng) and the number of unique molecules sequenced (R2=0.94). Importantly, we show that this low PCR bias allows OS-Seq to detect CNAs in Coriell and Horizon Diagnostic cell lines highly concordant to digital PCR detection (R2=0.96). Further, we present CNA calling on cell line DNA sonicated to 200 bp fragments at 10 ng DNA input, mimicking cell-free DNA. In addition to CNA detection, OS-Seq detects SNVs with a sensitivity of 92-97% and a specificity of 100% down to 5% VAF. In conclusion, the OS-Seq library preparation method relies on single stranded adapter ligation and in-solution target capture, which generates uniform coverage with minimal PCR requirement, resulting in highly sensitive CNA calling. Note: This abstract was not presented at the meeting. Citation Format: Anna Vilborg, Yosr Bouhlal, Ryan Koheler, Daniel Mendoza, Federico Goodsaid, Yannick Pouliot, Austin So, Francisco De La Vega, Hanlee Ji. A PCR-bias free capture-based library preparation platform permitting highly accurate and sensitive CNA detection in tumor molecular profiling and liquid biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4019. doi:10.1158/1538-7445.AM2017-4019
Type of Medium:
Online Resource
ISSN:
0008-5472
,
1538-7445
DOI:
10.1158/1538-7445.AM2017-4019
Language:
English
Publisher:
American Association for Cancer Research (AACR)
Publication Date:
2017
detail.hit.zdb_id:
2036785-5
detail.hit.zdb_id:
1432-1
detail.hit.zdb_id:
410466-3
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