Kapa dna library preparation kits

DNA isolation: DNA was isolated as previously described^{29 (link)}. Exome sequencing was performed by BGI (www.bgi.com) following standard protocols and by LGTC (www.lgtc.nl). At the LGTC, the generation of the library was performed using the KAPA DNA Library Preparation Kits for Illumina following the instruction of the manufacturer (Kapa BioSystems). Briefly, DNA was sheared by Covaris, followed by end repair, A-tailing and adapter ligation to both ends. DNA was amplified by ligation-mediated PCR (LM-PCR) and purified by Agencourt Ampure beads. The exome was enriched by hybridization of the amplified library with SureSelect Human all exome (v3) probes following the instructions of the manufacturer (Agilent Technologies). The enriched samples were quantified on Agilent Bioanalyzer and sequenced on Illumina HiSeq2000 using v3 reagents generating 100 bp long, pair-end tags. The sequencing was performed following the instructions of the manufacturer (Illumina).

Genomic DNA derived from the probands of the two kindreds (1ug/sample) was sheared to an average fragment size of 300bp. DNA libraries were prepared using the KAPA DNA library preparation kits for Illumina sequencing platforms. Exons were captured using a Roche NimbleGen SeqCap EZ library probe and the captured libraries were sequenced on a HiSeq2500. Processing of image files was performed using standard protocol. Alignment to Hg19 reference was performed using Burrows–Wheeler Aligner (BWA v0.7.15). Picard v2.5.0 was used to mark duplicate and low‐quality reads. Finally, GATK (Mckenna et al., 2010) was used for variant calling.