Reviewing the quality of reads by IGV
Raw sequencing data were reviewed for the detected variants using Integrative Genomic Viewer (IGV) software (https://software.broadinstitute.org/software/igv/) according to its instructions. Briefly, BAM files were imported to IGV and the quality of sequencing reads, coverage and number of the reads (depth) for the given variants were reviewed.

Variant Confirmation and Segregation Analysis

The reported positive findings in group A were reviewed with regard to the test setting (research or clinical), applied technique (NGS-based methods or Sanger sequencing), and the variant characteristics, to indicate whether further confirmatory tests were required. As a result, patients that were studied in a research setting, or had INDEL variants identified by WES, were investigated by Sanger verification. Although group B patients were studied by targeted-capture panel with high coverage depth, a subset of variants with depressed allele fraction (<30% of alternate allele), particularly deletion/insertion type variants, underwent additional validation with Sanger sequencing which was performed on an ABI3130 (Life Technologies) according to the standard procedures. Amplification primers were either selected from published papers (if available) or designed using Primer 3 software. Segregation analysis was carried out when additional family members were available and segregation data was helpful for classification and interpretation of the variants, especially variants with unknown clinical significance.

Complementary Sanger Sequencing

In case of no positive finding in target NGS, raw sequencing data were reviewed in each patient using IGV software, and low coverage regions were then sequenced by Sanger sequencing.