Abstract 3229: Validation of Illumina TruSeq Amplicon Cancer Panel with concordance testing using Ion AmpliSeq Cancer Panel and other methods.
Authors: Fang Peng , Yan Zhenyu , Liu Weihua , Darwanto Agus , Pelak Kim , Anoe Kim , Spittle Cynthia , Sankar Sabita , Galderisi Chad , Li Jin 
Published: August 2014

Abstract
Introduction: The TruSeq Amplicon Cancer Panel (TSACP), a highly multiplexed targeted resequencing assay for use on the Illumina MiSeq platform, is designed for detecting the hotspot mutations in 212 Regions of Interest (ROI) from 48 cancer related genes. Here we report our validation study on the reproducibility, sensitivity and the specificity of detecting single base substitutions and small indels by the TSACP.
Methods: We used well characterized cancer cell lines harboring clinically relevant variants as positive controls and HapMap samples NA12878 and NA19240 as wild type control samples. DNA from 8 cancer cell lines was serially diluted into the control DNA NA12878 for validating the Limit of Detection (LOD) of the TSACP assay. A total of 41 FFPE patient specimens representing a variety of cancer types were analyzed in a blinded fashion to evaluate the analytical sensitivity and specificity. DNA quality was assessed using a qPCR assay. With no gold standard available as a reference method to detect mutations with comparable sensitivity, concordance testing was performed using the Ion AmpliSeq Cancer Panel. Variants detected by both panels were considered as true positives. Variants that were only covered by one of the two panels were confirmed by a third method, either Sanger sequencing for variants with frequencies above 10% or a custom Ion TargetSeq Assay for variants with frequencies below 10%. Data were analyzed using MiSeq Reporter software and our proprietary analysis pipeline. In addition to reporting hotspots mutations, we also report “Critical Variants” such as non-synonymous coding mutations and splicing site mutations that fall within the ROI.
Results: 95% of our ROIs were sequenced at minimum of 0.2X normalized coverage. A cell line dilution study showed that the LOD of confirmed variants is 5%. DNA extracted from 4 of the 41 FFPE specimens failed the template QC by qPCR and failed in the subsequent sequencing run. A total of 124 unique critical variants, including single base substitution, single- or multi- base (up to 21bp) deletion, one- or two- base insertion, were identified in the cancer cell lines and 37 qualified FFPE samples. The intra-assay and inter-assay reproducibility was ≥96%. Using our proprietary analysis pipeline, the analytical sensitivity and specificity for the FFPE samples were both 99%. One false negative of TSACP was identified by the Ion AmpliSeq Cancer Panel and was further confirmed by Sanger sequencing. This allele drop-off occurred as a result of the capture probe falling on a SNP. Homopolymer indels in KIT and STK11 were accurately identified with the Illumina sequencing chemistry.
Conclusions: These studies demonstrate that the TSACP assay is highly specific and sensitive and is suitable for screening patient FFPE tumor specimens for a spectrum of clinically relevant somatic mutations.
Citation Format: Peng Fang, Zhenyu Yan, Weihua Liu, Agus Darwanto, Kim Pelak, Kim Anoe, Cynthia Spittle, Sabita Sankar, Chad Galderisi, Jin Li. Validation of Illumina TruSeq Amplicon Cancer Panel with concordance testing using Ion AmpliSeq Cancer Panel and other methods. [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 3229. doi:10.1158/1538-7445.AM2013-3229