DNA samples were submitted to the University of Washington Center for Mendelian Genomics (UW-CMG) where library construction, WES and analysis were performed. Briefly, sequencing libraries were generated for each DNA sample in an automated, 96-well plate format (Perkin-Elmer Janus II). Sample libraries were constructed from 1 μg of genomic DNA which underwent a series of shotgun library construction steps including: acoustic sonication (Covaris), end-repair, A-tailing ligation of unique sequencing adaptors, and PCR amplification. Sample libraries were hybridized to the Nimblegen SeqCap EZ v2.0 target (∼36.5 Mb) in multiplex for a period of 72 hrs. Captured DNA was then purified, PCR amplified, and normalized for sequencing.
Captured DNA was sequenced on Illumina HiSeq machines using paired-end sequencing. Raw sequence data in FASTQ format were aligned to the human genome reference hg19 using the BWA algorithm for the generation of BAM files9 (link). The quality of each sample was assessed for coverage (80% of sequenced target with ≥20× coverage and 90% of target with ≥8× coverage) and Ts/Tv ratios. Additionally, samples were quality controlled to confirm sex using PLINK (v1.90b2m) software, and estimations of kinship were corroborated with pedigrees using KINGv1.4.0 software.
Captured DNA was sequenced on Illumina HiSeq machines using paired-end sequencing. Raw sequence data in FASTQ format were aligned to the human genome reference hg19 using the BWA algorithm for the generation of BAM files9 (link). The quality of each sample was assessed for coverage (80% of sequenced target with ≥20× coverage and 90% of target with ≥8× coverage) and Ts/Tv ratios. Additionally, samples were quality controlled to confirm sex using PLINK (v1.90b2m) software, and estimations of kinship were corroborated with pedigrees using KINGv1.4.0 software.
