Nimblegen seqcap ez v2 kit

Exome sequencing was undertaken after obtaining appropriate informed consent. Genomic DNA was isolated from peripheral blood using the AUTOPURE LS Instrument (Gentra Systems). Cytogenetic microarray analysis was performed using the CytoScan HD Array (Affymetrix) according to the manufacturer's protocol. Copy number was assessed using Chromosome Analysis Suite software (Affymetrix). Whole exome sequencing was performed on DNA fragmented into 200- to 400-bp fragments using Adaptive Focused Acoustics (Covaris Inc) shearing according to the manufacturer's instructions. The exome was captured by Nimblegen SeqCap EZ V2 kit (Roche Nimblegen, Inc) in combination with Illumina paired end library preparation and 2 × 100 bp sequencing with at least 70x mean coverage. Downstream analyses included demultiplexing (CASAVA software; Illumina Inc), sequence quality control, capture quality control, single nucleotide polymorphism calling, and indel (insertions and deletions) calling using different software applications as described by Santen (11 (link)). Identified mutations were confirmed by Sanger sequencing (primer sequences available on request).

Genomic DNA was fragmented into 200–400 base pairs (bp) fragments using Covaris Adaptive Focused Acoustics shearing according to the manufacturer’s instructions (Covaris, Inc., Woburn, MA). Illumina TruSeq DNA Library preparation (Illumina, Inc., San Diego, CA) was performed on a Caliper Sciclone NGS workstation (Caliper Life Sciences, Hopkinton, MA), followed by exome capture using the Nimblegen SeqCap EZ V2 kit (Roche Nimblegen, Inc., Madison, WI). This capture targets 44 Mb of exonic regions covering 30,246 coding genes, 329,028 exons and 710 miRNAs. Paired-end 2 × 100 bp sequencing was performed at 6 samples per lane on Illumina HiSeq2000 sequencer using Illumina TruSeq V3, resulting in 6 Gb of sequencing data.