Approximately 3 µg of genomic DNA from each sample was processed through shearing to 150–200 bp pieces using a Covaris M220 Focused‐ultrasonicator (Covaris). An adaptor‐ligated library was constructed using a Paired‐end Sequencing Library prep Kit (Agilent Technologies). The design of the capture library was performed and enriched using an Agilent SureSelect XT Human All Exon V6 Kit (Agilent Technologies). Clusters were then generated using isothermal bridge amplification with an IllUmina cBot station, and the products were sequenced on an Illumina HiSeq2500 System (Illumina).
Base calling and sequence read quality assessments were evaluated using Illumina Sequence Control Software v4.0.4 with real‐time analysis (Illumina, Inc.). Sequencing reads were aligned to the reference human genome (GRCh37.3, SNP135) using NextGENe® (SoftGenetics LLC). All single‐nucleotide variants and insertion‐deletions were saved in a VCF format file and uploaded to the Ingenuity® Variant Analysis platform (Ingenuity System, Redwood City, CA, USA) for bioinformatics analysis and interpretation.
Base calling and sequence read quality assessments were evaluated using Illumina Sequence Control Software v4.0.4 with real‐time analysis (Illumina, Inc.). Sequencing reads were aligned to the reference human genome (GRCh37.3, SNP135) using NextGENe® (SoftGenetics LLC). All single‐nucleotide variants and insertion‐deletions were saved in a VCF format file and uploaded to the Ingenuity® Variant Analysis platform (Ingenuity System, Redwood City, CA, USA) for bioinformatics analysis and interpretation.