Targeted exome sequencing from a patient of HCM complicated with ERS
DNA extraction  Genomic DNA was extracted from peripheral blood lymphocytes by standard procedures using QIAamp DNA Bloodmini kits (Qiagen, Germany). Next, 1 ug genomic DNA was fragmented by Covaris sonicator (Covaris S2, USA) to sizes of 150-300 bp and then purified.   Target region capture  The blunt ends of the purified DNA fragments were then repaired, and A-tailing was added. The fragments were ligated overnight using standard Illumina paired- end (PE) adapter. The ligated products were then amplified through 4-cycle polymerase chain reactions (PCRs) using PE primers containing 8 bp index tags. The purified PCR products containing 3 ug DNA were hybridized to the GenCapTM probe (in solution) at 65°C for 22 hours using a PCR machine. The products were bound to a rotator for 1 hour at room temperature using Dynal Myone Streptavidin C1 magnetic beads (Invitrogen, USA), which had been activated beforehand, and the products were then washed with buffer according to the kit manual. The captured DNA libraries were amplified using 15-cycle PCRs, purified, and subsequently eluted in a 30 ul volume and subjected to Agilent 2100 Bioanalyzer and quantitative PCR to estimate the magnitude of enrichment.  Next Generation Sequencing  The final captured DNA libraries were sequenced using the Illumina HiSeq2000 Analyzers as PE 90 bp reads (following the manufacturer’s standard cluster generation and sequencing protocols), providing an average coverage depth for each sample of at least 100-fold.
Wuhan Asia Heart Hospital
YHS HCM sequencing