Lin M.-T.Mosier S.L.Thiess M.Beierl K.F.Debeljak M.LI-HUI TSENGChen G.Yegnasubramanian S.Ho H.Cope L.Wheelan S.J.Gocke C.D.Eshleman J.R.2022-03-102022-03-1020140002-9173https://www.scopus.com/inward/record.uri?eid=2-s2.0-84904502195&doi=10.1309%2fAJCPMWGWGO34EGOD&partnerID=40&md5=30eb3683a4b8dd5a5bd4911ed44a5d94https://scholars.lib.ntu.edu.tw/handle/123456789/597110Objectives: To validate next-generation sequencing (NGS) technology for clinical diagnosis and to determine appropriate read depth. Methods: We validated the KRAS, BRAF, and EGFR genes within the Ion AmpliSeq Cancer Hotspot Panel using the Ion Torrent Personal Genome Machine (Life Technologies, Carlsbad, CA). Results: We developed a statistical model to determine the read depth needed for a given percent tumor cellularity and number of functional genomes. Bottlenecking can result from too few input genomes. By using 16 formalin-fixed, paraffin-embedded (FFPE) cancer-free specimens and 118 cancer specimens with known mutation status, we validated the six traditional analytic performance characteristics recommended by the Next-Generation Sequencing: Standardization of Clinical Testing Working Group. Baseline noise is consistent with spontaneous and FFPE-induced C:G→T:A deamination mutations. Conclusions: Redundant bioinformatic pipelines are essential, since a single analysis pipeline gave false-negative and false-positive results. NGS is sufficiently robust for the clinical detection of gene mutations, with attention to potential artifacts. ? American Society for Clinical Pathology.BRAF; Deamination; EGFR; KRAS; Next-generation sequencing; Read depth; Validation[SDGs]SDG3[SDGs]SDG12B Raf kinase; epidermal growth factor receptor; formaldehyde; K ras protein; paraffin; article; bottleneck population; gene mutation; gene sequence; genetic procedures; genome; genotype; human; human cell; human tissue; major clinical study; next generation sequencing; point mutation; priority journal; pyrosequencing; single nucleotide polymorphism; BRAF; Deamination; EGFR; KRAS; Next-generation sequencing; Read depth; Validation; Deamination; High-Throughput Nucleotide Sequencing; Limit of Detection; Molecular Diagnostic Techniques; Mutation; Neoplasms; Paraffin Embedding; Proto-Oncogene Proteins; Proto-Oncogene Proteins B-raf; ras Proteins; Receptor, Epidermal Growth Factor; Reproducibility of Results; Sensitivity and Specificity; Tumor Markers, Biologicaljournal article10.1309/AJCPMWGWGO34EGOD248383312-s2.0-84904502195