Technical Validation of a Next-Generation Sequencing Assay for Detecting Actionable Mutations in Patients with Gastrointestinal Cancer

摘要: Targeted next-generation sequencing is becoming increasingly common as a clinical diagnostic and prognostic test for patient- tumor-specific genetic profiles well to optimally select targeted therapies. Here, we describe custom-developed, detecting single-nucleotide variants (SNVs) short insertions deletions (indels) in 93 genes related gastrointestinal cancer from routine formalin-fixed, paraffin-embedded specimens. We implemented validation strategy, based on the College of American Pathologists requirements, using reference DNA mixtures cell lines with known variants, which model broad range allele frequencies. Test sensitivity achieved >99% both SNVs indels, frequencies >10%, high specificity (97.4% 93.6% indels). further confirmed accuracies primary colorectal specimens characterized by alternative conventional technologies. Robust performance was observed specimens: 97.2% 99.2%. also intrarun inter-run reproducibility, low cross-contamination rate. Overall assessment line samples showed that our custom assay has consistent detection down 10% variant frequency.