Abstract 628: Determinants of quality of next-generation sequencing output from the strand-specific TruSight Tumor Sequencing Panel in a clinical diagnostic setting
Authors: Garg Swati , Sukhai Mahadeo A. , Thomas Mariam , Mah Michelle , Zhang Tong , Pugh Trevor , Kamel-Reid Suzanne , Stockley Tracey L. 
Published: August 2015

Abstract
The use of Next-Generation Sequencing (NGS) technologies is increasingly prevalent within diagnostic labs. As genomic regions are sequenced to greater depth in cancer diagnostics, it is critical to differentiate clinically actionable variants from artifacts arising from sequencing-errors, sample- processing or sample-age, and to identify samples that will be difficult to evaluate. We sought to determine whether strand-specific sequencing approaches, such as the TruSight Tumor Sequencing Panel (Illumina) could enable sample and variant triage in a clinical diagnostic settingTruSight Tumor Sequencing Panel allows for paired-end sequencing of individual strands of DNA and analyzing them either together (Paired) or separately (Pool A and Pool B). Variants identified in one pool, but not the other, are putative artifacts; variants identified in both pools are considered true calls. Combined analysis of both pools was performed in two ways: By summing variant calls across pools, and by informatically determining overlapping variant calls between pools. In a test cohort of 44 FFPE samples of varying age and tumor type, we assessed whether age of sample, strand bias, and fixation impacted the detection of high confidence variants using the TruSight Tumor Sequencing panel. Data were compared to the results of analysis of the same samples using the established Illumina TruSeq Amplicon Cancer Panel and/or Sanger Sequencing.Sample age, tumor cellularity, tumor type and template DNA quality were not found to be associated with quality of NGS output in our study. We also evaluated the overall transition/transversion (Ti/Tv) ratios for variants detected either uniquely in one pool or in combined analysis. Interestingly, for variants detected in both pools, the Ti/Tv ratio was 1.97, compared to 0.52-0.60 for those detected in only 1 pool (p &lt; 0.001). Strikingly, samples that sequenced successfully but gave inconclusive and difficult to interpret variant lists were associated with%G&gt;A:C&gt;T transition &gt; 62.5% and Ti/Tv ratios of &gt; 4.0 (p &lt; 0.001). G&gt;A:C&gt;T transitions were significantly over-represented in these samples. The overall%G&gt;A:C&gt;T transitions were equivalent (44-52%) in individual pools or in paired analysis. However, when inconclusive samples were accounted for, the%G&gt;A:C&gt;T transitions differed between the two analyses: 49.7% (paired) vs. 30.1-32.1% (individual pools). In summary, the Ti/Tv ratio can act as a critical determinant of variant call quality - Ti/Tv ratios ∼0.5 represent sequencing artifacts, while Ti/Tv ratios &gt; 4.0 are indicative of inconclusive sequencing output.We conclude that variant Ti/Tv ratio as well as%G&gt;A:C&gt;T transition in variants detected by the TruSight Tumor Sequencing Panel may be helpful evaluators of quality and clinical utility of sequencing output for FFPE tumor samples tested in a clinical diagnostic setting.
Citation Format: Swati Garg, Mahadeo A. Sukhai, Mariam Thomas, Michelle Mah, Tong Zhang, Trevor Pugh, Suzanne Kamel-Reid, Tracey L. Stockley. Determinants of quality of next-generation sequencing output from the strand-specific TruSight Tumor Sequencing Panel in a clinical diagnostic setting. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 628. doi:10.1158/1538-7445.AM2015-628