DOI: 10.18150/ajme6p
title: PEO1-OR cells acquire resistance through subclonal en-richment of BRCA2 secondary mutations that restore functional full-length protein - whole-exome sequencing.

description: Whole-exome sequencing (WES) was performed with the Agilent SureSelect Human All Exon V8 Kit (Agilent Technologies, Santa Clara, CA, USA[ŁB1] ) on PEO1 and PEO1-OR cell line tumor samples. Paired-end (2 × 150 bp) next-generation sequencing was performed on a NovaSeq 6000 Sequencing System (Illumina, San Diego, CA, USA[ŁB2] ) to obtain a mean coverage of more than 100×. Briefly, freshly thawed and recovered cells from passage 1 were seeded in 100 mm dishes (5 × 105 cells in 8 mL of culture medium) and cultured for four days (37 °C, 5% CO2) until reaching 80% confluency. Afterward, cells were harvested by trypsinization, and genomic DNA was isolated and purified using GenElute Mammalian Genomic DNA Miniprep Kits (Sigma-Aldrich) according to the manufacturer’s instructions. The quality, integrity, and purity of isolated DNA were evaluated using agarose gel electrophoresis and a spectrophotometer. Genomic DNA was stored in an ultrapure, molecular biology grade Tris-HCl solution (10 mM, pH 8.0) at –80 °C for a week until downstream analysis.Raw sequence data were aligned and annotated for individual samples to a reference sequence of the human genome (GRCh38). Duplicate reads were excluded from downstream analysis. Next, variants were called utilizing GATK’s Haplotype Caller and interpreted using Qiagen Clinical Insight Interpret Translational (QCI-IT) software. Briefly, SnpEff 4.3 prediction toolbox was used to annotate called variants and predict their effects on protein structure and function (gain of function, loss of function, normal function, or change of function). False-positive variants were filtered out using GATK’s Variant Filtration module.Thereafter, we restricted our analysis to the interpretation of single-nucleotide variants (SNVs) and short insertions and deletions (indels) in genes encoding studied proteins, i.e., p53 (TP53, NM_000546.6), ATR (ATR, NM_001184.4), CHK1 (CHEK1, NM_001114121.2), PARP1 (PARP1, NM_001618.4), PARG (PARG, NM_003631.5), BRCA1 (BRCA1, NM_007294.4), BRCA2 (BRCA2, NM_000059.4), MDR1 (ABCB1, NM_001348945.2), 53BP1 (TP53BP1, NM_005657.4), H2AX (H2AX, NM_002105.3), and RAD51 (RAD51, NM_002875.5), using the somatic workflow of QCI-IT software. To improve the high confidence of detected variants, we kept high-quality calls that passed upstream pipeline filtering and all of the confidence filters (call quality ≥30, genotype quality ≥30, and read depth ≥10). To keep only rare variants, any variant with a maximum population frequency &gt;5% in the gnomAD database was classified as benign and excluded from further interpretation (unless previously established as a pathogenic common variant), according to recommendations. High-quality variants were classified based on an automatically computed evidence-based categorization considering their pathogenicity (pathogenic, likely pathogenic, uncertain significance, likely benign, benign) and actionability in terms of therapeutic, diagnostic, and prognostic clinical significance (tier I—strong, tier II—potential, tier III—unknown, tier IV—benign or likely benign) according to the guidelines.