Agilent sureselectxt humanallexon v4 50 mb

Exome sequencing was performed on patient and both parents using the Agilent SureSelectXT HumanAllExon V4 (50 Mb) (Agilent Technologies). Briefly, 3 μg of genomic DNA was sheared in 130 μl of low TE buffer to a peak size of 150–200 bp using Covaris E220, and then purified with AmpPure XP beads to remove fragments less than 100 bp. The purified DNA fragments were then subjected to Agilent SureSelect Library preparation Kit, ILM, to be end-repaired, A-tailed, and ligated to indexing-specific paired-end adaptor. The adapter-ligated libraries were amplified for five cycles using Herculase II (Agilent Technologies). Amplified Pre-capture libraries (750 ng) were concentrated in 3 μl and hybridized to the target specific baits (SureSelectXT Human All Exon V4; Agilent Technologies) according to the manufacturer’s recommendations. Hybridized material was captured using streptavidin-coated beads (Invitrogen, Paisley, UK) and amplified for 10 cycles. Captured libraries were pooled in pairs and paired-end sequenced on one lane of the Illumina HiSeq 2000 at the Stanford Center for Genomics and Personalized Medicine.