Abstract 4218: Validation of the Ion AmpliSeq™ Comprehensive Cancer Panel (CCP) using castPCR™ technologies.
Authors: Li Kelly , Woo Cora , Mouanoutoua Mokang , Moy Bonnie , Langit Emanuel , Brzoska Pius , You Xiaoqing , Desai Sejal , Keys David , Stevens Junko , Kong Benjamin , Shannon Mark , Chen Shiaw-Min , Ruff David , Li Chieh-Yuan , Joun David , Casuga Iris , Bennett Robert 
Published: August 2014

Abstract
Somatic mutation has been implicated in many aspects of cancer such as susceptibility, diagnosis, prognosis, drug response and tumor progress. Detection of somatic mutation is of wide interest in cancer research. The rapid advances in next generation sequencing (NGS) technologies have transformed cancer research. For example, the Ion AmpliSeq™ technology enables the selective amplification of 10s to 1000s of target sequences in a single multiplexed PCR and meshes seamlessly with the Ion semiconductor sequencing platform. The Ion Ampliseq™ Comprehensive Cancer Panel (CCP) provides ready-access to hundreds of genes, making it ideal for broad targeted re-sequencing studies. Alternative technologies are in demand to validate the NGS data orthogonally and screen hundreds or more cancer samples to evaluate mutation patterns, prevalence and frequencies in population. Here, we have demonstrated the utility of TaqMan® Mutation Detection Assays using our competitive allele specific TaqMan® PCR (castPCR™) technology in validation of Ion Torrent sequencing data. In this study, we applied the Ion Ampliseq™ Comprehensive Cancer Panel (CCP) panel to NCI-60 cell lines (MCF-7, MDA-MB-231, DU-145, PC-3, SK-MEL- 28) derived from breast, prostate, and skin cancers on Ion Torrent PGM sequencer. We confirmed previously reported mutations in these cell lines and identified the mutations that were not reported before, including missense and non-coding mutations. We then selected a subset of mutation targets from the cancer panel for castPCR validation including the genes of KRAS. EGFR, BRAF, NRAS, PIK3CA, PTEN, KIT, TP53, and more. Since limited sample quantity has been a challenging issue for most cancer researchers, especially for those who are interested in testing multi-targets by qPCR, we developed a preamplification method to enrich the targets of interest prior to running castPCR assays. We compared the Cq values of on-targets and off-targets from preamplified samples and non-preamplified samples, and the data indicates that our preamplification strategy not only provides roughly100 fold target enrichment but also maintains specificity during the preamplification process. For mutation detection, we showed that the mutation data from castPCR™ and from Ion Torrent PGM sequencer share high concordance for any given mutations. The detailed comparison and analysis will be presented and discussed. Our results demonstrate that castPCR™ technology is a valuable validation tool for next generation sequencing. The combination of Ion Torrent sequencing and castPCR validation empowers cancer researchers to understand the roles that somatic mutation plays in cancer.
Citation Format: Kelly Li, Cora Woo, Mokang Mouanoutoua, Bonnie Moy, Emanuel Langit, Pius Brzoska, Xiaoqing You, Sejal Desai, David Keys, Junko Stevens, Benjamin Kong, Mark Shannon, Shiaw-Min Chen, David Ruff, Chieh-Yuan Li, David Joun, Iris Casuga, Robert Bennett. Validation of the Ion AmpliSeq™ Comprehensive Cancer Panel (CCP) using castPCR™ technologies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4218. doi:10.1158/1538-7445.AM2013-4218