Skip to main content

Validation of A Targeted Next-generation Sequencing Cancer Panel for Clinical Use

Friday, November 08, 2013 — Poster Session III

10:00 a.m. – 12:00 p.m.

FAES Academic Center (Upper-Level Terrace)




  • L. Xi
  • T.H. Pham
  • M.O. Evbuomwan
  • T. Pham
  • W.T. Navarro
  • M. Raffeld


Next-generation sequencing technologies offer new and sensitive approaches for detection of clinically relevant mutations. The AmpliSeq Cancer Hotspot Panel v2 (CHP2) targets 50 commonly mutated genes in cancer for subsequent sequencing on the Ion Personal Genome Machine. To assess the feasibility of incorporating this NGS approach for mutation detection into the clinical laboratory environment, we studied DNA from 34 FFPE tissue samples containing 33 known mutations, and from 3 normal hapmap samples. Variant analysis was performed using Ion variantCaller and IGV, and compared with allele quantification determined by pyrosequencing. An allele frequency limit of detection for SNVs of 2.5% and for indels of 10% was achieved. All 24 previously detected SNVs were identified by variantCaller, and additional SNVs, including 3 clinically important ones, were detected. Seven of the 9 indels were detected. The two indels missed by variantCaller, were seen at low frequency by IGV. No major variant was detected in the 3 normal samples. In conclusion, CHP2 shows promise for facilitating mutation detection in clinical FFPE samples. The assay shows good sensitivity and specificity, can be performed with as little as 10ng of FFPE DNA, and has a turnaround times of 5-7 days, all critical requirements for clinical use.

back to top