NGS-based Variant Profiling Protocol

NGS libraries were prepared as described previously [22 (link),23 (link)]. Briefly, 100 ng of fragmented genomic DNA was ligated with a specific barcode and pooled with 23 other sample libraries for a 24-plex run that was captured using the SeqCap EZ Choice Library system according to the manufacturer’s protocol (Roche NimbleGen, Inc., Madison, WI, USA). Captured libraries were diluted to 8 pM or 1.3 pM for sequencing with the MiSeq v2 or NextSeq v2.5 mid output kits, respectively (Illumina, San Diego, CA, USA). Sequencing reads were generated as 2 × 150 bp paired-end reads with post-sequencing file conversion to FASTQ for alignment with NextGene software version 2.4.2.3 (SoftGenetics, LLC, State College, PA, USA) using standard alignment settings. Variants were filtered at an allelic fraction of > 10% to minimise the impact of sequence artifacts and mutational burden and were classified by a clinical molecular geneticist based on the College of American Pathologists (CAP) and the American College of Medical Genetics and Genomics (ACMG) standards and guidelines for pathogenicity [24 (link),25 (link)]. For this study, all assessed Tier I/II variants (variants of strong and potential clinical significance (therapeutic, prognostic and diagnostic)) [24 (link)] and ACMG 1/2 variants (pathogenic or likely pathogenic variants) [25 (link)] were reported.