Ion Torrent Sequencing of Cancer Hotspot Mutations

NGS libraries were prepared according to the manufacturer’s protocol with the Ion AmpliSeq Library kit version 2.0 (Thermo Fisher Scientific). For DNA sequencing, 10 ng of DNA was subjected to multiplex PCR amplification with the use of an Ion AmpliSeq Cancer Hotspot Panel (CHPv2) primer pool (50 genes, 207 amplicons; Thermo Fisher Scientific). After multiplex PCR, Ion Xpress Barcode Adapters (Thermo Fisher Scientific) were ligated to the PCR products, which were then purified with the use of Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA). The purified libraries were pooled and then sequenced with the use of an Ion Torrent S5 instrument and Ion 550 Chip Kit (all from Thermo Fisher Scientific). DNA sequencing data were accessed through the Torrent Suite v.5.12 program (Thermo Fisher Scientific). Reads were aligned against the hg19 human reference genome, and variants were called with the use of Variant Call Format ver. 5.12. Raw variant calls were filtered with depth of coverage of <19 and were manually checked using the integrative genomics viewer (IGV; Broad Institute, Cambridge, MA, USA). Germline mutations were excluded with the use of the Human Genetic Variation Database (http://www.genome.med.kyoto-u.ac.jp/SnpDB) [51 (link)].