Evaluation of an integrated clinical workflow for targeted next-generation sequencing of low-quality tumor DNA using a 51-gene enrichment panel (BMC Medical Genomics)

Background: Improvements in both performance and cost for next-generation sequencing (NGS) have spurred its rapid adoption for clinical applications. We designed and optimized a pan-cancer target-enrichment panel for 51 well-established oncogenes and tumor suppressors, in conjunction with a bioinformatic pipeline informed by in-process controls and pre- and post-analytical quality control measures. Methods: The evaluation of this workflow consisted of sequencing mixtures of intact DNA to establish analytical sensitivity and precision, utilization of heuristics to identify systematic artifacts, titration studies of intact and FFPE samples for input optimization, and incorporation of orthogonal sequencing strategies to increase both positive predictive value and variant detection. We also used 128 FFPE samples to assess clinical accuracy and incorporated the previously described quantitative functional index (QFI) for sample qualification as part of detailing complete system performance. Results: We observed a concordance correlation coefficient of 0.99 between the observed versus expected percent variant at 250?ng input across 4 independent sequencing runs. A subset of the systematic variants were confirmed to be barely detectable on an independent sequencing platform (Wilcox signed-rank test p-value