Trusight oncology 500 panel

In SEV and SMC cohorts, BRCA mutation status was assessed in peripheral blood and tumor samples. Germline BRCA was tested using Sanger sequencing or next-generation sequencing (NGS). Sanger sequencing was performed on a 3730 DNA Analyzer with a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA), followed by analysis using Sequencher 5.3 software. NGS using a custom panel, including BRCA1 and BRCA2 genes, was performed in a proportion of patients on a NextSeq 550 instrument (Illumina) with 2 × 151 bp reads. Bioinformatic analysis was performed using the Burrows-Wheeler Aligner, Genome Analysis Toolkit, Ensembl Variant Effect Predictor, and a custom pipeline. Experienced geneticists made final interpretations. For tumor BRCA, genomic DNA was extracted for NGS of tumor samples using a Maxwell CSC DNA FFPE Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The products were sequenced using the Nestxeq550 System (Illumina) using the TruSight Oncology 500 panel (Illumina). For mutational analysis, FASTQ files were uploaded to the Illumina BaseSpace software (Illumina) for variant interpretation. Only variants in the coding regions, promoter regions, or splice variants with a minimum 3% of the reads and read depth of 250.

The Ion AmpliSeq Library Kit 2.0 (Thermo Fisher Scientific) was used to perform library preparation of 10 ng of genomic DNA using the Ion AmpliSeq Cancer Hotspot Panel v2 (Thermo Fisher Scientific, catalog number 4475346). The final library was quantified with the Ion Library Quantitation Kit (Thermo Fisher Scientific). Samples were multiplexed and amplified on Ion Spheres Particles with Ion 540 Kit-Chef and were sequenced using Ion 540 Chip (Thermo Fisher Scientific) with an adapted standard protocol^{56 (link)}.
Selected samples were processed using the TruSight Oncology 500 Panel (Illumina, catalog number 20028214). 100 base pairs were sequenced in paired-end mode on an Illumina NextSeq 550 machine. The raw data was demultiplexed and analyzed using the TruSight Oncology 500 v2.2 Local App Docker. Briefly, demultiplexed reads were alignment to the GRCh37 (hg19) genome using the Burrows-Wheeler Aligner, mapped reads were collapsed, re-aligned and stitched. Pisces software was used for somatic variant calling.

For amplicon-based targeted DNA-sequencing, 10–40 ng of DNA was isolated from stored snap frozen and archived FFPE tumor tissue or from cell lines using the DNeasy Blood & Tissue or the QIAamp DNA FFPE Tissue Kit and used for library preparation. Sequencing of cancer hotspot (CHP2v, ThermoFisher) or TruSight Oncology 500 panel (Illumina) libraries was performed with benchtop sequencers IonProton (Thermo fisher) or NextSeq2000 (Illumina) with 775X mean coverage (Supplementary Data 6). Sequencing results were analyzed with VariantCaller software and validated using databases such as Varsome, COSMIC, and the 1000Genomes project^{105 (link)}. Data of the latter enabled the separation of single nucleotide polymorphisms (SNPs) from mutations (single nucleotide variants, SNVs).