Targeted sequencing and variant calling

We then performed deep‐targeted exon sequencing of a prioritized panel of 41 genes that harbored polymorphisms reported to be associated with hypertension, the BP response to pharmacotherapy, and/or the BP response to PEH and exercise training (Ash et al. 2013a; Bruneau et al. 2015) using the Illumina TruSeq Custom Amplicon kit (Catalog# FC‐130‐1001, Illumina, San Diego, CA) (see Supplement Material for the prioritized panel of genes). The Illumina DesignStudio software was used to create probes for the generation of 1214 amplicons with a size range 225–275 bp. The TruSeq Custom Amplicon manifest file associated with this panel included Target ID, region, chromosome, and start and end hg19 reference coordinate positions. Sequencing libraries were prepared following the TruSeq Custom Amplicon Library Preparation Guide. DNA input mass for all libraries was 250 ng of DNA. Libraries were generated with dual indices (23 PCR cycles), followed by normalization and pooling. The library amplicon pool was sequenced using Illumina MiSeq version 2 reagents (250 paired‐end reads). 7.1 million pair‐end reads (6.8 million passing quality filter) were produced from the library amplicon pool. MiSeq Reporter Software (version 2.3.32), using the TruSeq Amplicon workflow was used to generate Fastq files and align reads to the hg19 human reference sequence using the Smith‐Waterman algorithm. Genome Analysis Toolkit (GATK) was use for variant calling (single‐nucleotide polymorphisms and small insertion/deletions) and the generation of variant calling files (VCF). For all further downstream analysis, a merged VCF was generated using VCFtools v 0.1.12b (Danecek et al. 2011) and custom R scripts (R v3.2.0). Only variants with FILTER = PASS were retained. For each defined amplicon target region, we calculated the total number of variants present per subject and each polymorphism's major and minor allele frequency for each subject.