WES and copy number variation (CNV) analysis

Human exome sequencing libraries were constructed using the Agilent SureSelect Human All Exon V5 kit (Agilent Technologies, Inc.), and amplicons were generated and sequenced with the Illumina HiSeq 2500 system (Illumina, Inc.). All procedures were performed according to the manufacturer's instructions. After sequencing, reads were aligned to an indexed human reference genome (GRCh37/hg19) with Burrows-Wheeler transformation 0.7.15-r1140(11). Duplicate reads were removed using Picard v.1.85 (http://picard.sourceforge.net) prior to further processing. Base recalibration and variant calling were performed using the Genome Analysis Toolkit v.2.3-4Lite (12). Finally, identified variants were saved in a variant call format. In addition, in the present study, there was an attempt to reveal CNVs with a read depth-based CNV detection method (13-16). The detection methods can be separated into four steps: Raw coverage normalization, correction for sample-specific coverage biases, CNV calling, and partially-mapped read analysis and all copy number variants were finally confirmed by Illumina Human Cyto SNP12 kit. Translational Genomics Expert (LifeMap Sciences, Inc.) was used for variant prioritization. Integrative Genomics Viewer (IGV; v.2.4.15; http://software.broadinstitute.org/software/igv/) was used to visualize WES data and assess the coverage of the exons.