Humanexome 12

Methods for collection and extraction of genomic DNA samples were described previously [21 (link)]. The EWASs were performed with the use of a HumanExome-12 v1.1 or v1.2 DNA Analysis BeadChip or Infinium Exome-24 v1.0 BeadChip (Illumina, San Diego, CA, USA). Exome array contains ~244,000 SNPs including common, low frequency, and rare variants located at whole exons. The GWAS makes use of high-throughput genotyping technologies that include up to 4.5 million markers for SNPs and copy number variations to examine their relation to clinical conditions or traits. The EWAS is a focus genotyping method that differs from the GWAS [23 (link)]. Detailed information of the exome arrays and methods of quality control were described previously [21 (link)]. A total of 41,843 SNPs passed quality control and was subjected to analysis.

ES analysis was performed at BG as previously described^{32 (link)}. Samples were also concurrently analyzed by SNP arrays (Illumina HumanExome-12 or CoreExome-24 array) for quality control of the ES data, as well as for detecting large copy-number variants (CNVs) and regions of absence of heterozygosity (AOH)^{33 (link),34 (link)}. Homozygous/hemizygous deletions were also analyzed using an in-house developed pipeline based on exome read-depth analysis as previously described^{35 (link)}. The ES-targeted regions cover >23,000 genes for capture design (VCRome by NimbleGen^®), including both coding and untranslated region exons. The mean coverage of target bases was >100×, and >95% of target bases were covered at >20×^{32 (link)}. PCR amplification and Sanger sequencing was performed to verify all candidate variants in the probands according to standard procedures. Of note, reanalysis of ES data for individuals who had their first ES analysis prior to January 2020 was performed as described recently^{36 (link)} to evaluate for the presence of other potentially causative variants. No other potential molecular diagnoses contributed by other loci were identified by the reanalyses.