Exome sequencing and analysis
MUTATION
Genomic DNA was extracted from peripheral blood samples and submitted for whole exome sequencing at the Regeneron Genetics Center (RGC). Briefly, 1 μg of genomic DNA was fragmented and prepared for exome capture with a custom reagent kit from Kapa Biosystems. Samples were captured using the NimbleGen SeqCap VCRome 2.1 exome target design and sequenced using 75-bp paired-end sequencing on an Illumina HiSeq 2500 with v.4 chemistry. Following sequencing, data were processed using a cloud-based pipeline developed at the RGC that uses DNAnexus and AWS to run standard tools for sample-level data production and analysis. Sequence reads were mapped and aligned to the GRCh37/hg19 human genome reference assembly using BWA-mem. Single-nucleotide polymorphisms and INDEL variants and genotypes were called using GATK’s HaplotypeCaller. Standard quality-control filters were applied to called variants. Passing variants were classified, annotated and analyzed using an RGC-implemented Mendelian analysis pipeline to evaluate their potential functional effects. Variants were annotated for their observed frequencies in population control databases such as dbSNP, the 1000 Genomes Project, the Exome Aggregation Consortium Database and internal RGC databases to filter out common polymorphisms and high frequency, likely benign variants. Algorithms for bioinformatic prediction of functional effects of variants (LRT, Poly-phen2, SIFT, CADD and Mutation Taster), along with conservation scores, were incorporated as part of the annotation process of variants and used to inform on the potential deleteriousness of identified candidate variants. Individuals in this study were screened for variants in a list of 28 genes compiled for their reported associations with triglyceride or lipid levels.Screened genes were 