Whole blood

Next-generation sequencing was performed at the Dr. von Hauner Children's Hospital NGS facility. Genomic DNA was isolated from whole blood (Qiagen) for the generation of whole-exome libraries using the SureSelect XT Human All Exon V6 + UTR kit (Agilent Technologies). Barcoded libraries were sequenced with a NextSeq 500 platform (Illumina) to an average coverage depth of 90x. Bioinformatics analysis used Burrows-Wheeler Aligner (BWA 0.7.15), Genome Analysis ToolKit (GATK 3.6) and Variant Effect Predictor (VEP 89). The frequency filtering used allele frequencies from public (e.g. ExAC, GnomAD and GME) and in house databases. The potentially causative variants were confirmed by Sanger sequencing for the patient and informative family members.

For all study subjects, genomic DNA was extracted from whole blood (Qiagen, Netherlands) and validated in terms of quality and quantity using standard techniques. The TruSight Cardio sequencing kit from illumina (illumina Inc) was used to prepare libraries that are enriched for the coding and flanking intronic boundaries of 174 genes known to be involved in inherited cardiac conditions (ICCs) (Pua et al., 2016 (link)) (Table S1). DNA sequencing was performed on illumina Miseq instrument (illumina Inc) using either v2 or v3 chemistry kit to produce 300‐bp paired‐end sequencing reads.

Genotyping was performed using the Illumina CardioMetaboChip v1.0 (Illumina, San Diego, CA) following the manufacturer’s protocol [21 (link), 22 (link)] in a total of 11,582 subjects using DNA purified by column from whole blood (Qiagen, Valencia, CA). The chip has been described elsewhere [4 (link)]. Calling of genotypes was performed using the Illumina Genome Studio software and both samples and SNPs were removed with call rates less than 98%.