Ion Ampliseq Cancer Hotspot Panel v2 Protocol

The Ion Ampliseq Cancer Hotspot Panel v2 on the Ion Torrent platform was performed to detect 2790 mutations in 50 oncogenes and tumour suppressor genes^{16 (link),30 (link)}. In brief, 10 ng of genomic DNAs extracted from 55 frozen tumour samples were used to construct barcoded DNA libraries utilizing the Ion Ampliseq Library Kit 2.0 (Thermo Fisher Scientific). The obtained libraries were optimized using Ion Library Equalizer Kit (Thermo Fisher Scientific), and then sequenced using an Ion Personal Genome Machine or Ion S5XL platform (Thermo Fisher Scientific). The sequencing reads were aligned to the reference genome build hg19, GRCh37, and converted into BAM files using Ion Torrent Suite software (Thermo Fisher Scientific). Sequence variants were called using Ion Reporter 5.0 (Thermo Fisher Scientific), according to the manufacturer’s instructions. The average sequencing depth reached at least 1500-fold per sample.