Whole-exome sequencing and data analysis
We extracted genomic DNA from tissue specimens and blood samples using the QIAamp DNA mini kit according to the manufacturer’s protocol (Qiagen, Valencia, CA, USA). The concentration and quality of DNA were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). WES was performed using the SureSelect Human All Exon V5 Target Enrichment kit (Agilent Technologies, Santa Clara, CA, USA) and processed on the HiSeq 2500 platform (Illumina, San Diego, CA, USA). The size and quantity of the libraries were checked using a 2100 Bioanalyzer (Agilent Technologies).
Sequence reads were aligned to the human reference genome hg19 using the Burrows-Wheeler Aligner–MEM algorithm [7]. The alignments were refined using the Genome Analysis Tool Kit (Broad Institute), and duplicate or low-quality reads were excluded [8]. Somatic mutations were called using Mutect and Strelka with default settings by comparing the sequences of tumor samples with those of matched normal samples [9, 10]. Only mutations showing a total read depth of greater than 30 in normal samples were considered for further analysis.