Clinical validation of KRAS, BRAF, and EGFR mutation detection using next-generation sequencing

Ming Tseh Lin, Stacy L. Mosier, Michele Thiess, Katie F. Beierl, Marija Debeljak, Li Hui Tseng, Guoli Chen, Srinivasan Yegnasubramanian, Hao Ho, Leslie Cope, Sarah J. Wheelan, Christopher D. Gocke, James R. Eshleman

Research output: Contribution to journalArticlepeer-review

126 Scopus citations


Objectives: 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.

Original languageEnglish (US)
Pages (from-to)856-866
Number of pages11
JournalAmerican journal of clinical pathology
Issue number6
StatePublished - Jun 2014

All Science Journal Classification (ASJC) codes

  • Pathology and Forensic Medicine


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