Whole exome sequencing (WES) analysis of two colorectal patients with metastasis

Six specimens from 2 colorectal cancerpatients, including 2 triples of primary MT, matched LM and PN, were available for CMA and WES analysis. DNA and cRNAs were hybridized to Affymetrix CytoScan HD Array as described [Wang, 2014]. Genomic DNA was enriched for exonic regions using the SureSelect Biotinylated RNA Library (BAITS). The sequences of captured libraries were generated as 90-bp pair-end reads on an Illumina Hiseq2000. Sequencing reads processing and mapping to the reference GRCh37/hg19 human genome assembly using the Burrows-Wheeler Aligner (BWA) was performed as described [O'Roak, 2012]. Further processing, including duplicate removal, local realignment and base quality recalibration, single nucleotide variants (SNVs) and indel calling and filtering was performed using the Genome Analysis Toolkit (GATK) [McKenna A, 2010], SAM [Li, 2009], and Picard tools (http://picard.sourceforge.net). Then, filters were applied to obtain variant results of higher confidence and annotation and classification were performed using ANNOVAR [Wang, 2010]. The variant collection was excluded from positions reported in the 1000 Genomes Project and dbSNP. Only genes that harbored heterozygous variants that were truncating mutations or splice-site variants were selected for further analyses.