Targeted Sequencing for Variant Analysis

Genomic DNA was extracted from peripheral blood using a QIAamp DNA Blood Mini Kit (Qiagen, Venlo, The Netherlands). The amount of input DNA was approximately 500 ng. DNA was fragmented into segments between 150 and 250 bp using the Bioruptor^® Pico sonication system (Diagenode, Liege, Belgium), end-repaired, and ligated to Illumina adapters (Illumina, San Diego, CA, USA) and indices. Sequencing libraries were hybridized with capture probes (Celemic, Seoul, Korea). The enriched DNA was then amplified, and clusters were generated and sequenced on a NextSeq 550 instrument (Illumina) with 2 × 151 bp reads [17 (link)]. Pathogenicity interpretations of the variants were performed according to the 2015 American College of Medical Genetics and Genomics guidelines by professional medical geneticists, using evidence from variant type assessments, population allele frequency, prediction algorithm results, and searches within databases such as ClinVar.