Dragen bio it platform version 3

Whole exome sequencing was performed using the Twist Human Core Exome Plus Kit (Twist Bioscience, San Francisco, CA, USA) as the gene capture kit, and the enriched libraries were sequenced on a NovaSeq 6000 sequencing machine (Illumina, San Diego, CA, USA). For each sample, paired end reads (2×100 bp) were obtained and processed. The Illumina Dragen Bio-IT Platform version 3.8 was used to align reads to the human reference genome (hg38) based on the Smith-Waterman algorithm,^{12 (link)} as well as to call variants based on the GATK variant caller version 3.7.^{13 (link)} Additional variants were called with Freebayes version 1.2.0.^{14 (link)} Variant annotation was performed using KGG-Seq version 1.2.^{15 (link)} Further annotation and filtration steps were performed by in-house scripts using various additional public and private datasets such as the Human Gene Mutation Database,^{16 (link)} ClinVar,^{17 (link)} gnomAD^{18 (link)} and the Sheba Medical Center's database of all variants from previously sequenced samples. Final variant analysis was only performed on rare mutations with an allele frequency of <0.05 in gnomAD and the in-house database, and that were annotated as protein changing and protein-damaging based on KGG-Seq, in addition to all variants that were previously reported as pathogenic or likely pathogenic in the HGMD or ClinVar datasets.

Libraries for tumor and corresponding normal tissues were prepared with the Agilent SureSelect XT HS2 system and incorporation of unique molecular indices (UMI). Target enrichment was performed with the Clinical Exome v2 bait set. In brief, tumor DNA extracted from FFPE tissue and DNA from total blood was manually sheared on a Covaris ME220 ultrasonicator to fragment sizes of 200–500 bp. 80–100 ng DNA were used for end repair and adaptor ligation according to the manufacturer's instructions. Hybridization was performed overnight followed by bead purification. Final libraries were qualified on an Agilent D1000HS tape and fluorimetrically quantified using QuBit instrument (Thermo Fisher). Four tumor/normal pairs (ratio 4:1) were pooled and sequenced together on a NovaSeq6000 SP flow cell (Illumina) using 2 × 101 bp reads. Data analysis was performed on a local Illumina DRAGEN Bio-IT platform version 3.8 using somatic tumor-normal and germline only workflows using the genome assembly GRCh37.