Abstract 3182: Single tube, ultra-multiplexed PCR with Ion AmpliSeq™ technology for amplicon resequencing on the Ion Personal Genome Machine™ System
Authors: Roma Gianluca 
Published: August 2012

Abstract
Abstract: Translational and clinical cancer research require rapid approaches that are compatible with small input amounts of challenging research samples such as FFPE tissue. By overcoming known barriers of multiplex PCR, the Ion AmpliSeq™ technology introduces a groundbreaking workflow enabling the rapid sequencing of hundreds of known mutations with low allele frequency with detection down to 5%. Utilizing low input DNA, this single-tube, single day workflow is as simple as setting up a PCR reaction. The Ion AmpliSeq™ Cancer Panel is a fixed-content panel that matches the specific needs of clinical and translational researchers. Rapid and parallel assessment of 739 key somatic and germline mutations in 46 gene targets is enabled. Simple workflow allows library preparation in just 3.5 hours. Comparisons will be presented on results from tumor/normal pairs on FFPE DNA samples from various cancers samples including lung, thyroid and colon. The Ion AmpliSeq™ Custom Assays allows cancer researchers the ability to select targets of choice to accommodate between 10 kb and 1 Mb of cumulative human genome coverage. Using the web-based Torrent Assay Designer, researchers can quickly submit gene names and/or gene coordinates up to 768 amplicon per pool and multiple pools can be combined to run on a single Ion chip allowing up 12,288 amplicons to be assayed at once. Results will be presented on a 768 amplicon panel being used to test several tumor vs normal paired samples. Method: Genomic DNA was extracted from FFPE samples using the PureLink® Genomic DNA Mini Kit. Samples were processed according to the Ion AmpliSeq™ Library Preparation User Guide. The extracted DNA was quantitated with the Qubit® Fluorometer. Ten ng of DNA was amplified via PCR using a master mix of 190 or 768 cancer primer pairs. The amplicons were then purified and phosphorylated. Template preparation automated on the Ion OneTouch™ System. After template preparation the amplicon library from each sample was loaded onto an Ion 314™ chip and sequenced in 90 minute on the Ion PGM™ sequencer. The Torrent Suite Software was used for variant calling via the AmpliSeq Cancer Variant plug-in. Results & Conclusions: We detected 100% of the known somatic mutations and detected no false-positives. The Ion AmpliSeq™ technology has shown itself to be the fastest and simplest approach to generating amplicon libraries of targeted regions of interest. The Ion AmpliSeq™ Cancer Panel is capable of detection 739 known Cosmic mutations across 46 key cancer genes in a single day with high sensitivity and high accuracy. The Ion AmpliSeq™ Custom assays enable quick custom assay design turn-around and accurate data generation for fast project completion. Details on further fixed-content cancer panels will be presented.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3182. doi:1538-7445.AM2012-3182