Abstract 4215: Rapid, inexpensive and accurate detection of rare variants by targeted sequencing.
Authors: Dressman Devin , Nishimura Stefanie , Scafe Charles , Fu Yutao , Fanti Ellen , Kotler Lev , Panchapakesa Vaish , Orphe Wendell , Hansen Sherry , Hapshe Nick , Fergus Carl , Lagier Erin , Wei Eric , Lui Grace , Nallapareddy Reddy , Chen Xiaohui , Liu Zhitong , Namsaraev Eugeni 
Published: August 2014

Abstract
Next generation sequencing technology is quickly becoming the most rapid and accurate method of determining mutation profiles of patient's tumor samples. While whole genome or exome sequencing profiles are the most comprehensive methods for complete exploration of these tumors, these protocols typically require large amounts of DNA, are expensive, don't get deep coverage, and require extensive time for library and sample preparation. Ion Torrent has developed new methodology for looking at 10’s to 1000’s of genomic targets with the Ion AmpliSeq™ technology. The recommended input of 10ng of DNA is suitable for work with FFPE or fine needle biopsy samples, and has been demonstrated on samples with less than 1ng of DNA. In a 10 hour day this protocol can take isolated DNA through the library and sample prep as well sequencing with variant calls, making it an incredibly rapid method for determining sequence variants within targeted regions. There are currently two commercially available cancer-specific amplicon panels: the Comprehensive Cancer Panel (CCP) uses ∼16,000 primer pairs covering the exons of 409 tumor suppressor genes and oncogenes frequently cited and mutated in cancers, and the Cancer Hotspot Panel v2 (CHP2) uses 207 primer pairs in 50 clinically actionable genes to detect over 2800 mutation hotspots. We recently measured the sensitivity of SNP detection levels with the CCP and CHP2 panels on a 90:10 (reference:SNP) admixed sample and were able to detect SNP proportions of 2.0%(p&lt;0.005) with CHP2 and 2.0% (p&lt;0.001) with CCP. The CHP2 amplicons yielded coverages of &gt;2000X for a single sample on a 314 chip and &gt;3000X for 6 barcoded samples on a 318 chip. Comparatively, the average base coverage of the CCP was &gt;280x and over 95% of the CCP amplicons were covered at &gt;50X. We ran the CCP in quadruplicate on 318 chips at two separate facilities and the CHP2 was run barcoded on 318 chips and single samples on 314 chips also in quadruplicate at two separate facilities and all of the chips show very equivalent numbers for SNP detection and coverages. These results demonstrate the consistent nature of targeted sequencing using a next gen platform. Going from isolated DNA to variant calling in as little as 10 hrs, this method is extremely fast and efficient which will be critical in future diagnostic settings.
Citation Format: Devin Dressman, Stefanie Nishimura, Charles Scafe, Yutao Fu, Ellen Fanti, Lev Kotler, Vaish Panchapakesa, Wendell Orphe, Sherry Hansen, Nick Hapshe, Carl Fergus, Erin Lagier, Eric Wei, Grace Lui, Reddy Nallapareddy, Xiaohui Chen, Zhitong Liu, Eugeni Namsaraev. Rapid, inexpensive and accurate detection of rare variants by targeted sequencing. [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 4215. doi:10.1158/1538-7445.AM2013-4215