Validation of Targeted Next Generation Sequencing on Ion Torrent Platform for Clinical Testing of Solid Tumors
Somak Roy, Mary Beth Durso, Yuri Nikiforov, Marina Nikiforova. University of Pittsburgh, Pittsburgh, PA
Background: Next generation sequencing technologies have unleashed the possibility of multiplex sequence variation (SV) detection without significant increase in cost. In this study, we evaluated the performance of Ion AmpliSeqTM Cancer Panel on Ion Torrent PGMTM (Life Technologies, San Francisco, CA) for testing of solid tumors in a clinical molecular diagnostic laboratory.
Design: Total of 92 tumors, 8 cell lines and 3 normal tissues were tested for SVs in 46 genes using the Ion AmpliSeqTM Cancer Panel. The validation set included 55 tumors with documented SVs in the KRAS, NRAS, HRAS, BRAF, RET, KIT, EGFR, PDGFR, TP53 genes and 37 tumors without SVs. Analysis was performed using the 316 and 318 chips with up to 10 barcoded samples per chip, AmpliSeq v.2.0 chemistry, One Touch 200v2 DL Template kit and Ion PGM Sequencing 200 kit. Data were analyzed using Torrent Suite v2.2, Variant Caller Plugin v2.2.3-31149 and NextGENe® (Softgenetics, State College, PA).
Results: Only 10ng of DNA was sufficient for analysis of all samples (40 frozen, 51 FFPE, 4 FNAs and 8 cell lines) for SVs in 46 genes. An average of 536,660 (189,802–1,334,101) mapped sequence reads per barcoded sample was achieved with the 72bp mean read length, 2,783 mean reads for hot spot coverage, and 92% uniformity of coverage. One hundred percent (53/53) single nucleotide variants, 71% (5/7) deletions and 22% (2/9) insertions were detected with an overall sensitivity of 88%. Manual review of the sequence reads in IGV v.2.1.24 (Broad Institute, Cambridge, MA) revealed 1 deletion and 4 insertion not detected by Variant Caller. In addition to known SVs, novel SVs were detected in the PIK3CA, TP53, MET, ATM, and KDR genes (confirmed by Sanger sequencing). Multiple low-frequency SVs were detected across all samples and were frequently attributed to read errors in homopolymer regions. A limit of SVs detection was 5-10% depending on coverage depth and turnaround time was 72 hours.
Conclusions: Targeted sequencing using the AmpliSeqTM Cancer panel on Ion TorrentTM PGM allows testing for a broad spectrum of SVs with minimum amount of DNA and rapid turnaround time. Although the sensitivity for detection of single nucleotide variants was 100%, use of alternate protocols is recommended to circumvent the limitations in insertion/deletion detection and homopolymer-associated read errors for clinical use of this platform.
Category: Special Category - Pan-genomic/Pan-proteomic approaches to Cancer
Tuesday, March 5, 2013 9:30 AM
Poster Session III # 224, Tuesday Morning