Whole Genome Sequencing Protocol for Cancer

Libraries for WGS were prepared using Illumina TruSeq PCR-free reagents. WGS samples were sequenced using 2 × 150-bp paired-end reads, on a HiSeqX v2.5 (patient 1) or NovaSeq 6000 (patients 2 and 3) instrument (Illumina). DNA sequence data were processed with Sarek, following the GATK best-practice recommendations^{39 (link)}, on UPPMAX Clusters at Uppsala University (https://www.uppmax.uu.se/resources/systems/the-bianca-cluster/). In brief, the steps run were quality control of the FASTQ files using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/), alignment of short reads to the human reference genome sequence (GRCh38/hg38) using bwa-mem with the ALT-aware option turned on⁴⁰ , sorting of reads and marking of PCR duplicates with GATK MarkDuplicates and base quality score recalibration and joint realignment of reads around indels using GATK tools (https://github.com/broadinstitute/gatk). Tumour CNV profiles were generated using Control-FREEC^{41 (link)}. The matched normal sample was used to call somatic CNVs.