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, S Yegnasubramanian, Hao Ho, Leslie Cope, Sarah Wheelan, Christopher Gocke, James Eshleman

Research output: Contribution to journalArticle

Abstract

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
Volume141
Issue number6
DOIs
StatePublished - 2014

Fingerprint

Mutation
Genome
Paraffin
Formaldehyde
Neoplasms
Ions
Technology
erbB-1 Genes
Deamination
Statistical Models
Computational Biology
Artifacts
Noise
Genes

Keywords

  • BRAF
  • Deamination
  • EGFR
  • KRAS
  • Next-generation sequencing
  • Read depth
  • Validation

ASJC Scopus subject areas

  • Pathology and Forensic Medicine

Cite this

Clinical validation of KRAS, BRAF, and EGFR mutation detection using next-generation sequencing. / Lin, Ming-Tseh; Mosier, Stacy L.; Thiess, Michele; Beierl, Katie F.; Debeljak, Marija; Tseng, Li Hui; Chen, Guoli; Yegnasubramanian, S; Ho, Hao; Cope, Leslie; Wheelan, Sarah; Gocke, Christopher; Eshleman, James.

In: American Journal of Clinical Pathology, Vol. 141, No. 6, 2014, p. 856-866.

Research output: Contribution to journalArticle

Lin, Ming-Tseh ; Mosier, Stacy L. ; Thiess, Michele ; Beierl, Katie F. ; Debeljak, Marija ; Tseng, Li Hui ; Chen, Guoli ; Yegnasubramanian, S ; Ho, Hao ; Cope, Leslie ; Wheelan, Sarah ; Gocke, Christopher ; Eshleman, James. / Clinical validation of KRAS, BRAF, and EGFR mutation detection using next-generation sequencing. In: American Journal of Clinical Pathology. 2014 ; Vol. 141, No. 6. pp. 856-866.
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