Gentra puregene blood kit plus

DNA isolation and exome sequencing was conducted as previously described.^{4 (link)} Briefly, peripheral blood was collected from patients and stored at 4° C until genomic DNA was extracted using the Gentra Puregene Blood Kit Plus (Qiagen, 158489). For some parental samples, DNA was extracted from saliva using prepIT-L2P DNA isolation kit (DNA Genotek, PT-L2P-45). For exome sequencing, three to six micrograms of DNA were used for library preparation and sequencing at the Beijing Genomics Institute (BGI) collaborative genome center at CHOP (BGI@CHOP). Exome capture was performed using the Agilent SureSelect Whole Exome, version 4 kit (51 MB target size), and 100 base pair paired end sequencing was performed on Illumina HiSeq 2500 sequencing machines (4 samples per lane in high throughput mode). Exome sequencing performance was monitored using the following quality metrics: mean coverage depth (≥100X), fraction of target covered with at least 20X (>95%), mapping rate (>95%), and base calling accuracy (Q20 > 85%). The ES analysis strategy combined proband-only ES with proband and parental sample (where available) Sanger sequencing validation for all reported variants.