blood wes
DNA extraction and quality assessmentThe blood sample was thawed at room temperature no more than 30min and mixed by inversion for a few seconds. 1ml Red Blood Cell Lysis Buffer(10mmol/L Tris-HCl,0.5mmol/L MgCl2, 1% TritonX-100) was added into the 2.0ml tube with 1ml blood and mixed by vortex for 15s. The tube was Centrifuged at 16700g for 10 min at room temperature and carefully removed the supernatant with a pipette tip. 900ul Cell lysis buffer(10mmol/L Tris-HCl,400mmol/L NaCl 2mmol/L EDTANa2 0.8mol/L Guanidine Hydrochloride), 50L 20mg/mL Proteinase K and 50L 10% SDS was added into the tube and Mixed by vortex for 15s then Incubating at 56C in a water incubator for 60 min. After cooling to room temperature, 1ml Phenol / Chloroform / isoamylalcohol (25:24:1) was added and Gently inverted to mix liquid no obvious boundaries then Centrifuging at 16700g for 10 min at room temperature. Transferring the supernatant to a new 1.5mL tube and adding 2/3th volume of supernatant of isopropyl alcohol (add 1/10th volume of 3M sodium acetate if necessary), then it was inverted at least 3 times and placed at -20C for 2 hours for precipitation. To remove the supernatant, the tube was centrifuged at 16700g for 10 minutes getting the DNA pellet and it was washed with 1 ml 75% ethanol. Re-suspend the pellet by centrifuging at 16700g for 5 minutes at room temperature and Completely remove the supernatant. Air-dry the DNA pellet for a few minutes and add 25L~100L of TE Buffer to dissolve the DNA.DNAs Concentration was detected by Qubit Fluorometer. Sample integrity and purity were detected by Agarose Gel Electrophoresis.Library construction and whole exome sequencing1g genomic DNA was randomly fragmented by Covaris to an average size of 250-300bp. Fragmented DNAs were tested by agarose gel electrophoresis and purified with the AxyPrep Mag PCR clean up Kit. The selected fragments were through end-repair, 3 adenylated, adapters-ligation, PCR Amplifying and the PCR products were recovered by the AxyPrep Mag PCR clean up Kit. Certain amount of PCR products was taken for hybridization with BGI Hybridization and Wash kits. The captured DNA fragments from previous step was amplified and the PCR products were purified with the AxyPrep Mag PCR clean up Kit. Then Library was qualified by the Agilent Technologies 2100 bioanalyzer and ABI StepOnePlus Real-Time PCR System. The qualified libraries were sequenced pair end on the Hiseq4000/Xten/Novaseq System (BGI-shenzhen, china).