Validation of a Customized Bioinformatics Pipeline for a Clinical Next-Generation Sequencing Test Targeting Solid Tumor-Associated Variants

Bioinformatic analysis is an integral and critical part of clinical next-generation sequencing. It is especially challenging for some pipelines to consistently identify insertions and deletions. We present the validation of an open source tumor amplicon pipeline (OTA-pipeline) for clinical next-generation sequencing targeting solid tumor-associated variants. Raw data generated from 557 TruSight Tumor 26 samples and in silico data were analyzed by the OTA-pipeline and legacy pipeline and compared. Discrepant results were confirmed by orthogonal methods. The OTA-pipeline reported 22 variants that were not detected by the previously validated pipeline, including seven synonymous or intronic single-nucleotide variants, five single-nucleotide variants at frequency <5%, one insertion, and nine deletions. Variant allele frequencies reported by the two pipelines were highly concordant, although a few significant discrepancies were present. Analysis of in silico FASTQ files demonstrated a higher sensitivity of detecting complex insertions and deletions with the OTA-pipeline. The higher sensitivity came at a cost, because false-positive calls were increased in difficult-to-sequence regions. However, these calls were all flagged by our strand bias filter, distinguishing them from true variants. Our validation process provides a model for laboratories that want to establish an in-house bioinformatics pipeline for clinical next-generation sequencing.