Ion torrent s5 instrument

For the primary tumor tissues, mutations in the 409 cancer-related genes were analyzed using an Ion AmpliSeq Library Kit 2.0 and Ion AmpliSeq™ Comprehensive Cancer Panel (CCP) (Thermo Fisher Scientific, Waltham, MA, USA). Tumor DNA was extracted using All Prep DNA/RNA Mini Kit (Qiagen, Hilden, Germany) and 40 ng of DNA were subjected to multiplex PCR amplification. Then, the Ion Xpress Barcode Adapters (Thermo Fisher Scientific) were ligated to the PCR products, which were then purified with Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA). The purified libraries were pooled, and then sequenced with the Ion Torrent S5 instrument and Ion 550 Chip Kit (both from Thermo Fisher Scientific). DNA sequencing data were accessed through the Torrent Suite v.5.10 program (Thermo Fisher Scientific). Reads were aligned against the hg19 human reference genome, and variants were called using Variant Call Format ver. 5.10. Raw variant calls were filtered by quality score <100, depth coverage <19, and were manually checked using the integrative genomics viewer (IGV; Broad Institute, Cambridge, MA, USA). Germline mutations were excluded using the Human Genetic Variation Database (http://www.genome.med.kyoto-u.ac.jp/SnpDB) and the Exome Aggregation Consortium database.

A targeted DNA library comprising approximately 1.2 Mb of the coding regions of 409 genes for panel sequencing was constructed using an Ion AmpliSeq CCP (Thermo Fisher Scientific) in accordance with the manufacturer’s recommended protocol. Briefly, 40 ng of DNA were subjected to multiplex PCR amplification using an Ion AmpliSeq Library Kit 2.0 and an Ion AmpliSeq CCP (Thermo Fisher Scientific), covering all exons in 409 genes. After multiplex PCR, Ion Xpress Barcode Adapters (Thermo Fisher Scientific) were ligated to the PCR products, which were then purified using Agencourt AMPure XP beads (Beckman Coulter). The purified libraries were pooled and then sequenced using an Ion Torrent S5 instrument and an Ion 550 Chip Kit (all from Thermo Fisher Scientific). DNA sequencing data were accessed through the Torrent Suite ver. 5.10 program (Thermo Fisher Scientific). Reads were aligned against the hg19 human reference genome, and variants were called using Variant Caller ver. 5.10. The raw variant calls were filtered with a depth of coverage of <19, quality score of <100, and synonymous variants and were manually checked using the integrative genomics viewer (IGV, Broad Institute). Germline mutations were excluded using the Genome Aggregation Database (gnomAD [>0.1%], ExAC [>0.1%]) and the Human Genetic Variation Database.