Validation of a next-generation sequencing assay for clinical molecular oncology

Catherine E. Cottrell, Hussam Al-Kateb, Andrew J. Bredemeyer, Eric J. Duncavage, David H. Spencer, Haley J. Abel, Christina M. Lockwood, Ian S. Hagemann, Stephanie M. O'Guin, Lauren C. Burcea, Christopher S. Sawyer, Dayna M. Oschwald, Jennifer L. Stratman, Dorie A. Sher, Mark R. Johnson, Justin T. Brown, Paul F. Cliften, Bijoy George, Leslie D. McIntosh, Savita ShrivastavaTudung T. Nguyen, Jacqueline E. Payton, Mark A. Watson, Seth D. Crosby, Richard D. Head, Robi D. Mitra, Rakesh Nagarajan, Shashikant Kulkarni, Karen Seibert, Herbert W. Virgin IV, Jeffrey Milbrandt, John D. Pfeifer

Research output: Contribution to journalArticlepeer-review

159 Scopus citations


Currently, oncology testing includes molecular studies and cytogenetic analysis to detect genetic aberrations of clinical significance. Next-generation sequencing (NGS) allows rapid analysis of multiple genes for clinically actionable somatic variants. The WUCaMP assay uses targeted capture for NGS analysis of 25 cancer-associated genes to detect mutations at actionable loci. We present clinical validation of the assay and a detailed framework for design and validation of similar clinical assays. Deep sequencing of 78 tumor specimens (≥1000× average unique coverage across the capture region) achieved high sensitivity for detecting somatic variants at low allele fraction (AF). Validation revealed sensitivities and specificities of 100% for detection of single-nucleotide variants (SNVs) within coding regions, compared with SNP array sequence data (95% CI = 83.4-100.0 for sensitivity and 94.2-100.0 for specificity) or whole-genome sequencing (95% CI = 89.1-100.0 for sensitivity and 99.9-100.0 for specificity) of HapMap samples. Sensitivity for detecting variants at an observed 10% AF was 100% (95% CI = 93.2-100.0) in HapMap mixes. Analysis of 15 masked specimens harboring clinically reported variants yielded concordant calls for 13/13 variants at AF of ≥15%. The WUCaMP assay is a robust and sensitive method to detect somatic variants of clinical significance in molecular oncology laboratories, with reduced time and cost of genetic analysis allowing for strategic patient management.

Original languageEnglish
Pages (from-to)89-105
Number of pages17
JournalJournal of Molecular Diagnostics
Issue number1
StatePublished - Jan 2014


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