Next Generation Sequencing in Clinical Environment for Cancer Therapy
Boin Lee, Yoon-La Choi, Ha Young Park, Young Hye Ko. Samsung Medical Centre, Sungkyunkwan University School of Medicine, Seoul, Korea
Background: Clinical practice for targeted therapy requires rapid approaches that are compatible with small input amounts of nucleic acids derived from challenging samples including formalin-fixed paraffin-embedded (FFPE) tissues. Furthermore, the number of targetable genes is increasing and multiplex testing with small amount of sample is required. By overcoming known barriers of multiplex PCR, the Ion AmpliSeqTM technology introduces a groundbreaking workflow enabling the rapid sequencing of hundreds of known mutations with low-frequency allele detection. The Ion AmpliSeq Cancer Panel consists of 190 amplicons and surveys 790 known cosmic mutations in approximately 20 kb of sequence from 46 genes including EGFR, KRAS and BRAF. This kit is ideally suited for both fresh and FFPE samples, requiring only 10ng of DNA per reaction.
Design: Library preparation was performed according to the manufacturer's protocol. Library constructions of the amplicons and subsequent enrichment of the sequencing beads were performed using the OneTouch system. Sequencing was done on the 316 chip with 10 megabases capacity using the Ion Torrent PGM (Life Technologies) as per the manufacturer's protocol. Data analysis, including alignment to human reference genome and base calling, was done using built-in software. We tested 60 solid cancers including bladder, gastric, breast, colon, kidney, lung and pancreatic cancer using fresh or FFPE samples.
Results: The turn-around-time was about 1 week from sample preparation to final analyzed report. Some of results were validated by Sanger sequencing. False positive calling by homopolymer deletion was identified. Mean read coverage of amplicons at the known cosmic hotspot mutation sites was calculated, and we noticed that each amplicon showed various read coverage (5X to ∼11,000X).Many amplicons of FFPE had slightly less coverage than fresh samples. Most cases showed one or two mutation among 46 genes, most common gene are as follows; TP53, KRAS, and PIK3CA.
Conclusions: The Ion AmpliSeq Cancer Panel contains most actionable genes that have been tested in clinical lab with reasonable turn-around time and high accuracy. Furthermore, this is applied to small samples of FFPE which is most common sample type in clinical environment.
Category: Special Category - Pan-genomic/Pan-proteomic approaches to Cancer
Tuesday, March 5, 2013 9:30 AM
Poster Session III # 235, Tuesday Morning