1. Combining semiconductor-based sequencing with amplicon-based cancer gene panels: A rapid next-gen approach to clinical cancer genotyping. Christopher L. Corless, Tanaya Neff, Michael C. Heinrich, Carol Beadling Knight Cancer Institute, Oregon Health & Science University, and Portland Veteran's Affairs Medical Center, Portland, Oregon Background: Bringing next-gen sequencing into clinical (CLIA- licensed) laboratories is an important step in the advancement of personalized cancer care. We have validated a new semiconductor –based sequencing approach (Ion Torrent PGM) in combination with amplicon-based library preparations. Using FFPE-derived samples of tumor and normal DNA, the performance of two panels has been examined in detail: 1) the AmpliSeq Cancer Panel (available from Ion Torrent); 2) a custom panel designed for genotyping GI stromal tumors (GISTs). Designed to detect hotspot mutations across 46 genes. Samples tested: 45 FFPE solid tumors previously genotyped on Sequenom MassArray system, including : - 53 point mutations - 19 in/dels (4 – 63 bp) Input DNA: 10 ng 190 primer pairs (1 tube) 13.3 kb of target sequence Barcoded samples 4 samples per 316 chip Results On-target reads: 95% Average read length:76 bp Average read depth: 2000 (range 8 – 4,948) All 53 point mutations were identified. All 19 in/dels were visible in the sequence, but most were not accurately identified by the Torrent Suite software (version 2.0). Using a cut-off of 8% mutant allele, the sensitivity for known mutations was 100% and the specificity was 92%. There were 27 new non-synonymous mutations with >8% mutant allele frequency detected in regions not covered by the mass spectrometry-based panel, including 24 point mutations and 3 deletions of 2 bp; all 27 were confirmed by Sanger sequence analysis. Conclusions: Combining solid-state sequencing with a highly multiplexed PCR method for library construction is a rapid (48 hr) approach for next-gen sequencing of clinical cancer samples. The protocol requires very little DNA (10-20 ng). Indeed, two DNA samples from laser-captured tumor yielded excellent data with the AmpliSeq Cancer Panel. The process is highly scalable and larger, cancer-specific amplicon panels are in development. Automated identification of in/dels remains a challenge in next-gen sequencing output. Recent improvements in the library preparation protocol and in the variant caller software are leading to further reductions in turn-around time and the number of background variants. Both the Cancer Gene Panel and the GIST Panel are now available in our CLIA-licensed/CAP-accredited laboratory. Acknowledgements: We are grateful for the help of our collaborators at Ion Torrent, including Katherine Rhodes, Michael Thornton, John Leamon and Mark Andersen. ABL1FGFR3NRAS AKT1FLT3PDGFRA ALKGNASPIK3CA APCHNF1APTEN ATMHRASPTPN11 BRAFIDH1RB1 CDH1JAK2RET CDKN2AJAK3SMAD4 CSF1RKDRSMARCB1 CTNNB1KITSMO EGFRKRASSRC ERBB2METSTK11 ERBB4MLH1TP53 FBXW7MPLVHL FGFR1NOTCH1 FGFR2NPM1 AKT1KRASSDHA AKT2MAP2K1SDHAF1 AKT3NF1SDHAF2 ATMNRASSDHB BRAFPDGFRASDHC CDKN2APIK3CASDHD HRASPTENTP53 KITPTPN11 AmpliSeq Cancer Gene Panel (Ion Torrent) Reads per amplicon (Mean + standard dev.) Reads per amplicon (Mean + standard dev.) Comparison of allele ratios: MassArray vs AmpliSeq 53 Point Mutations R=0.92, P<0.0001 Custom GI Stromal Tumor (GIST) Panel AmpliSeq Cancer Gene Panel (Ion Torrent) Input DNA: 20 ng 477 primer pairs (2 tubes) Amplicons tiled for whole gene coverage 125 to 175 bp amplicons 49.1 kb of target sequence Barcoded samples Primers were designed with the assistance of collaborators at Ion Torrent using the Ion AmpliSeq designer software v1.0. Samples tested: 19 FFPE GISTs that were previously shown to be negative for KIT and PDGFRA mutations. 8 barcoded samples run on a single 318 chip, yielding an average of 337,000 reads per sample 11 barcoded samples run on 316 chips (@ 4 per chip), yielding an average of 392,000 reads per sample Results On-target reads: 88% Average read length: 86 bp Average read depth: 695 (range 11 – 1,848) An NRAS Q61L mutation was confirmed in one case; this mutation is novel in GIST Knight Diagnostic Laboratories