TY - JOUR T1 - A Robust Targeted Sequencing Approach for Low Input and Variable Quality DNA from Clinical Samples JF - bioRxiv DO - 10.1101/123117 SP - 123117 AU - Austin So AU - Anna Vilborg AU - Yosr Bouhlal AU - Ryan T. Koheler AU - Susan M. Grimes AU - Yannick Pouliot AU - Daniel Mendoza AU - Federico Goodsaid AU - Mike Lucero AU - Francisco M. De La Vega AU - Hanlee P. Ji. Y1 - 2017/01/01 UR - http://biorxiv.org/content/early/2017/04/01/123117.abstract N2 - Next-generation sequencing is being adopted as a diagnostic test to identify actionable mutations in cancer patient samples. However, clinical samples such as formalin-fixed, paraffin-embedded specimens frequently provide low quantities of degraded, poor quality DNA. To overcome these issues, many sequencing assays rely on extensive PCR amplification leading to an accumulation of bias and artifacts. Thus, there is a need for a targeted sequencing assay that performs well with DNA of low quality and quantity without relying on extensive PCR amplification. We evaluated the performance of a targeted sequencing assay based on Oligonucleotide Selective Sequencing. This assay enables on to sequence and call variants from low amounts of damaged DNA. This assay utilizes a repair process developed to sequence clinical FFPE samples, followed by adaptor ligation to single stranded DNA and a primer-based capture technique. This approach generates sequence libraries of high fidelity without relying heavily on PCR amplification, and facilitates the assessment of copy number alterations across the target regions. Using an assay designed to capture the exons of a panel of 130 actionable cancer genes, we obtain an on-target rate of >50% and high uniformity across targeted regions at starting input DNA amounts of 10ng per sample. We demonstrate the performance of this targeted sequencing assay using a series of reference DNA samples, and in variant identification from low quality DNA samples originating from different tissue types. ER -