Using Digital PCR to Verify NGS for Cancer Research | ASHG 2015

Marion Laig of Thermo Fisher Scientific answers questions about her poster that was presented the 2015 American Society of Human Genetics annual meeting.

Abstract
We identified mutations in eleven cell free (cf) DNA samples by next generation sequencing (NGS) using the Ion AmpliSeq™ Colon & Lung Cancer Research Panel and the Ion PGM™ System. Since detection of low frequency mutant alleles may not always be called confidently in NGS, we verified results by rare mutation analysis using digital PCR on the QuantStudio™ 3D Digital PCR System as an independent method. We show that frequencies detected are consistent for both methods for low frequency mutant alleles at and below 1%.

Next generation sequencing (NGS) is becoming an increasingly important analysis tool for the discovery of biomarkers in cancer research and to study tumor heterogeneity in samples. Although NGS is an excellent discovery tool, detection of low frequency mutant alleles may not always be called confidently due to an inherent low error rate of the DNA polymerase used (fidelity). It is therefore beneficial to verify detection of rare alleles with an independent
method. In this study, we set out to confirm NGS sensitivity and accuracy detecting low frequency mutations using rare mutation analysis by digital PCR as an independent technology. We present data showing consistency in low level mutation detection in cf DNA samples between two methods: NGS and Rare Mutation Analysis using digital PCR