Combining semiconductor-based sequencing with amplicon-based cancer gene panels: A rapid next-gen approach to clinical cancer genotyping.

Abstract

10591 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 sequencing approach on the Ion Torrent (IT) PGM using the AmpliSeq Cancer Panel, which covers hotspot regions across 46 commonly mutated cancer genes. Methods: The AmpliSeq panel is comprised of 190 primer pairs that are co-amplified in a single tube to generate amplicons for sequencing. In our testing only 10ng of input DNA was used. Initial PCR was for 20 cycles, after which the amplicons were ligated with sequencing/barcode adapters, amplified for an additional 7 cycles, and then subjected to emulsion PCR. The resulting nanospheres were sequenced on an IT 316 chip. Results: We sequenced 44 samples of FFPE-derived tumor DNA that were previously genotyped on a mass spectroscopy (MS)-based panel. Samples were barcoded and sequenced in batches of 4, yielding an average of 2034 reads per amplicon (range: 95-5162) and an average read length of 76bp. Overall, 95.4% of reads were on target, and 79% of reads were AQ20 or better; 95% of the 190 amplicons had over 500 reads. All 42 known point mutations were accurately identified by the variant caller software. Seventeen in/dels from 4 to 63 bp in length were at least partially visible upon manual inspection of the read alignments, but some were not accurately called by the software. In addition, 22 new mutations were identified in gene regions not covered on our MS-based panel. We demonstrated sensitivity to the level of 5% mutant allele, and the correlation between allelic ratios measured by MS and IT sequencing was excellent (r2=0.81). Two DNA samples from laser-captured tumor worked well with the AmpliSeq Panel. 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 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.