Rader_HHDL_Targeted_DNA_Sequencing
Individuals of European ancestry with phenotypically extreme HDL-C levels were selected to undergo targeted sequencing.  Using customized hybrid capture (Agilent) baits, we targeted and enriched for the sequencing of the exons of the ~990 genes located within 300kb of each of the 95 loci with significant associations (P < 5 x 10-8) with plasma lipid levels identified by the Global Lipids Genetics Consortium (Teslovich TM et al. Nature. 2010 Aug 5;466(7307):707-13.).  Libraries of synthetic 200-mer oligodeoxynucleotides were obtained from Agilent Technologies. The pool for exon capture consisted of 22,000 oligonucleotides of the sequence 5′-ATCGCACCAGCGTGTN170CACTGCGGCTCCTCA-3′ with N170 indicating the target-specific bait sequences. Baits were tiled along exons without gaps or overlaps starting at the left-most coding base in the strand of the reference genome sequence shown in the UCSC genome browser (that is, 5′ to 3′ or 3′ to 5′ along the coding sequence, depending on the orientation of the gene) and adding additional 170-mers until all coding bases were covered. The synthetic oligonucleotides for regional capture consisted of 10,000 200-mers that targeted 4,409 distinct 170-mer sequences, of which 3,227 were represented twice (that is, the sequence above plus its reverse complement) and 1,182 were represented thrice. For baits designed to capture a predefined set of targets, we chose the minimal set of unique olignonucleotides and added additional copies (alternating between reverse complements and the original plus strands) until the maximum capacity of the synthetic oligonucleotide array (currently up to 55,000) was reached.   We performed next generation sequencing using Illumina Genome Analyzers.  Targeted sequencing was performed using standard Illumina adaptor sequences of fragment libraries at the Broad Institute using the method of Gnirke and colleagues (Gnirke et al. Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing. Nat Biotechnol. 2009 Feb;27(2):182-9. doi: 10.1038/nbt.1523.). Overall, the coverage was 30X for 77% of the 649,375 targeted bases sequenced. Base pairs were called and sequencing reads were aligned to the human genome reference GRCh37 (hg19), and sequencing metrics were processed using the Picard pipeline with an output of Binary Alignment Map (BAM) files. The Genome Analysis Toolkit Unified Genotyper was used to genotype all variant sites, calculate initial quality control metrics, and filter based on these values, and variants were annotated using SnpEFF. Standard individual-level quality control measures led to the exclusion of 9 individuals with a high heterozygosity rate (a measure of contamination), and 7 individuals due to hybrid selection failure. Standard variant-level quality control measures across all individuals led to the following overlapping exclusions: 8,707 variants due to standard Genome Analysis Toolkit filters across all batches (part of Broad Institute pipeline), 440 variants with a low call rate (<95%), 2,786 variants due to a low average depth of coverage (<8), and 4,436 variants due to a batch minor allele frequency >0.05. Finally, 3 additional individuals were excluded due to an excessively high number of called mutations, determined qualitatively.