Whole exome-sequencing of homo sapiens: blood genomic DNA
The gDNA met the sequencing requirements of the purity (optical density (OD)260/280>1.8) and concentration (50 ng/ml) of each sample was fragmentized into 100-800 bp pieces with a peak size of ~250 bp using NEBNext dsDNA Fragmentase (New England Biolabs, Ipswich, MA, USA), followed by end-repairing, dA-Tailing and adaptor ligation using the NEBNext DNA Library Prep Reagent Set from Illumina (New England Biolabs, Ipswich, MA, USA). The adaptor-ligated DNA fragments were fractionated by 2% agarose gel electrophoresis and fragments of the desired size (300400 bp) were excised. The extracted DNA was amplified in 10 PCR cycles using PE primers (Illumina, San Diego, CA, USA) and Phusion DNA polymerase (New England Biolabs, Ipswich, MA, USA). The PCR products were then subjected to exome sequence capture using the Illumina Truseq Exome Enrichment kit V3, which contains a 31.3 Mb CCDS (97.2% of the US National Center for Biotechnology Information CCDS Database) region across ~20,794 genes within 62 Mb of coding exons, according to the manufacturers manual (Illumina, San Diego, CA, USA). The enriched elution was amplified in 10 PCR cycles using PE primers and Phusion DNA polymerase. The amplicons were size-checked and quantitated using a BioAnalyzer 2100, and then subjected to 2  100 bp paired-end massively parallel sequencing using a Hiseq2000 Sequencing System (Illumina, San Diego, CA, USA).