Seqcap ez choice library system

Custom sequence capture probes were designed using the SeqCap EZ Choice Library system (Roche NimbleGen, Inc., Madison, WI, United States). The design included enrichment for all coding exons as well as 20 bp of the flanking intronic regions. Normally, 100 ng of genomic DNA was used for library preparations. Library preparation and target capturing sequencing steps were performed as previously described (Schenkel et al., 2016 (link); Kerkhof et al., 2017 (link)).

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.