title: Whole-exome sequencing; homo sapiens; blood samples: Sample P301
design: Genomic DNA quality is determined by estimating the A260/A280 and A260/A230 ratios by nanodrop, both of which should be > 1.8, and by 1% agarose gel electrophoresis. High quality DNA will migrate as a single high molecular weight band. 1 µg of genomic DNA is sheared to a mean fragment length of about 140 bp using focused acoustic energy (Covaris E210). Fragmented DNA samples are transferred to a 96-well plate and library construction is completed using a liquid handling robot. Magnetic AMPure XP beads (Beckman Coulter) are used to purify the sheared DNA samples and remain with the sample throughout library construction. Following each process step, DNA is selectively precipitated by weight and re-bound to the beads through the addition of a 20% polyethylene glycol, 2.5 M NaCl solution.  Following fragmentation, T4 DNA polymerase and T4 polynucleotide kinase blunt end and phosphorylate the fragments. The large Klenow fragment then adds a single adenine residue to the 3' end of each fragment and custom adapters (IDT) are ligated using T4 DNA ligase.  Adapter-ligated DNA fragments are then PCR amplified using custom-made primers (IDT).  During PCR, a unique 6 base index is inserted at both ends of each DNA fragment. Equal amounts of each sample are pooled prior to capture. The dried sample is reconstituted according the manufacturer's protocol (Roche/Nimblegen), heat-denatured, and mixed with biotinylated DNA probes produced by Roche/Nimblegen (SeqCap EZ Design). Hybridizations are performed at 47°C for 16 hours. Once the capture is complete the samples are mixed with streptavidin-coated beads and washed with a series of stringent buffers to remove non-specifically bound DNA fragments. The captured fragments are PCR amplified and purified with AMPure XP beads. Sample concentrations are normalized to 10 nM and loaded onto Illumina Rapid or High-output flow cells at a concentration that yields 150-250 million passing filter clusters per lane. Samples are sequenced using 100 bp paired-end sequencing on an Illumina HiSeq 4000 according to Illumina protocols.
attributes.tag: alignment_software
attributes.value: bwa mem