Seqcap ez hypercap workflow

BCR-ABL1 KD amplicon libraries were prepared using the Nextera XT DNA Library Prep Kit (cat. number FC-131-1096, Illumina, San Diego, CA, USA).
The DNA custom-designed NGS panel of 33 genes often mutated in myeloid malignancies (Supplementary Table S2) was analyzed using SeqCap EZ HyperCap Workflow (Roche, San Diego, CA, USA).
Sequence analysis and mutation identification were performed using NextGENe software (SoftGenetics, State College, PA, USA).

Extracted genomic DNA samples were placed into three groups based on genomic profile size ranges of <700 bp, 700–1100 bp, and 1100–2500 bp, and were subject to fragmentation to 180–220 bp using a Covaris E220 Series Focused-ultrasonicator (Covaris, Inc., Woburn, MA) with recommended settings and treatment times of 20, 50, and 60 s, respectively. Libraries were then prepared with the SeqCap EZ HyperCap workflow according to manufacturer’s protocol (Roche NimbleGen, Inc., Madison, WI, USA) and captured as a 24-plex pool with a custom target design that enriched for all coding exons, as well as 20 bp of the 5′ and 3′ flanking intronic regions for 37 hereditary cancer genes. Four captured libraries (96 samples) were diluted to 4 nM each and pooled for sequencing on the NextSeq according to the manufacturer’s protocol (Illumina, San Diego, CA, USA). The libraries were sequenced using the NextSeq version 2 mid output reagent kit to generate 2 × 150 bp paired-end reads. Post-sequencing file conversion generated FASTQ files for sequence alignment with the NextGene software version 2.4.1 (SoftGenetics, LLC, State College, PA, USA) using the recommended settings. The identified variants were filtered by an allelic fraction >10% and were assessed based on CAP guidelines for pathogenicity.