Exome Sequencing and SNP Genotyping from Blood/Saliva

Genomic DNA (gDNA) was extracted from blood (or rarely, saliva) samples of consenting study participants using Gentra Puregene kit (Qiagen, MD). DNA concentration was measured in a NanoDrop spectrophotometer or by fluorescence using Qubit (Thermo Fisher Scientific, MA). gDNA from study participants were sent to our collaborator, Regeneron Genetics Center LLC (Tarrytown, NY), for exome sequencing and SNP genotyping. Due to poor DNA quality, or sex discrepancy, or contamination, 55 samples were excluded from the analysis. Exons in the remaining samples were captured using the IDT xGen Exome Research Panel v1.0 (Integrated DNA Technologies, Coralville, IA) and sequenced at >30X coverage on the Illumina HiSeq2500 platform (Illumina, San Diego, CA). Raw reads were mapped to GRCh38 using Burrows-Wheeler Alignment Tool (BWA) [54 (link)] and variants were called using the Genome Annotation Toolkit (GATK) Best Practices pipeline (https://software.broadinstitute.org/gatk/best-practices/). GATK’s Variant Quality Score Recalibration (VQSR) procedure was performed to extract superior quality variants. All Mendelian errors, genotypes with GQ < 20, DP < 10 and AB < 0.25/ > 0.75, and variants with >2% missing calls were excluded.