Whole Exome Sequencing and Variant Analysis

We extracted DNA from whole blood and quantified it with a fluorometer (Qubit; Life Technologies Corporation, Carlsbad, CA, USA). The exome was captured and enriched from genomic DNA using an exome enrichment kit (Nextera; Illumina Inc., San Diego, CA, USA), representing 62 Mbp of the human genome (hg19 build). Sequencing was performed with a high-resolution optical imaging system (HiScan; Illumina Inc.).
Fragments were aligned to the hg19 reference genome through the use of Burrows-Wheeler alignment [41 (link)], and variant calling was performed with a software package for the analysis of sequence data (Genome Analysis Toolkit; Broad Institute, Cambridge, MA, USA) [42 (link)–44 ]. A total of 800 informative single nucleotide polymorphisms were analyzed with the whole-genome association analysis toolset (PLINK; http://pngu.mgh.harvard.edu/~purcell/plink/) to confirm the familial relationship between samples [45 (link)]. For genotype calls (in all members of the quartet, we used only reads with a Phred score ≥ 30 [46 (link)], bases with ≥ 20 reads, and a genotype quality score ≥ 20 [33 (link)].