Targeted Exome Sequencing of Cancer Genes

Genomics DNA (250 ng) from each tissue was sheared in a Covaris S220 ultrasonicator (Covaris, Woburn MA, USA) and used for the construction of a library with CancerSCAN™ probes and a SureSelect XT reagent kit, HSQ (Agilent Technologies) according to the manufacturer's protocol. This panel was designed to enrich exons of 83 genes [34 (link)] covering 366.2 kb of the human genome. After enriched exome libraries were multiplexed, the libraries were sequenced using the 100-bp paired-end mode of the TruSeq Rapid PE Cluster Kit and TruSeq Rapid SBS kit on the Illumina HiSeq 2500 sequencing platform (Illumina Inc., San Diego, CA, USA). The DNA sequence data were aligned to the human genome reference (hg19) using the MEM algorithm in BWA 0.7.5 [35 (link)]. Duplicate read removal was performed using Picard v.193 and SAMTOOLS v0.1.18 [36 (link)]. Local alignment was optimized using the Genome Analysis Toolkit (GATK) v3.1-1 [37 (link)]. We also used BaseRecalibrator from GATK for base recalibration based on known single nucleotide polymorphisms (SNPs) and indels from Mills, dbSNP138, and 1000G gold standard, 1000G phase1 and Omni 2.5. Sequencing coverage is shown in Supplementary Table 5.