TruSight™ oncology 500assay

Forty (40) ng of DNA was quantified with the Qubit dsDNA HS Assay (Thermo Fisher Scientific) on the Qubit 2.0 Fluorometer (Thermo Fisher Scientific) and then sheared using a Covaris E220 Focused-ultrasonicator (Woburn, MA, USA) and the 8 microTUBE–50 Strip AFA Fiber V2 following the manufacturer’s instructions. Treatment time was optimized for FFPE material. The treatment settings were as follows: peak incident power (W): 75; duty factor: 15%; cycles per burst: 500; treatment time (s): 360; temperature (°C): 7; and water level: 6. For DNA library preparation and enrichment, the TruSight™ Oncology 500 Kit (Illumina) was used following the manufacturer’s instructions. Post-enriched libraries were quantified, pooled, and sequenced on a NextSeq 500 (Illumina Inc., San Diego, CA, USA). The quality of the NextSeq 500 (Illumina) sequencing runs was assessed with the Illumina Sequencing Analysis Viewer (Illumina). Sequencing data were analyzed with the TruSight^™ Oncology 500 Local App Version 1.3.0.39 (Illumina), a comprehensive tumor profiling assay designed to identify known and emerging tumor biomarkers, including small variants, splice variants, and fusions. The reads were aligned to the reference genome (GRCh37/hg19) using Burrows − Wheeler Aligner-MEM (BWA-MEM) (Li 2013). Poorly mapped reads with a mapping quality (MAPQ) below 20 were removed using Samtools version 1.3.1(Li et al. 2009). Somatic mutations including single-nucleotide variants (SNV) and small insertions and deletions (INDELs) were detected by the Pisces and Psara (Dunn et al. 2019). The rest of pipeline are as follows: CRAFT for copy number variation, TmbRaider for TMB, Hubble for MSI, STAR for RNA alignment, and Manta for fusion calling (Pestinger et al. 2020). Outputs of data, exported from The TSO 500 pipeline (Pestinger et al. 2020) were annotated with Ensembl Variant Effect Predictor (VEP) Annotation Engine, with information from the databases, such as dbSNP, gnomAD genome and exome, 1000 genomes, ClinVar, COSMIC, RefSeq, and Ensembl. The processed genomic alterations were categorized with four-tier system by American Society of Clinical Oncology and College of American Pathologists (Li et al. 2017), annotated with proper reference. The following criteria were used to filter our less significant variants and possible germline variants: (i) variants < 5% allele frequency and < 100 × read depth at the variant were excluded; (ii) variants previously reported to be benign or likely benign in the ClinVar archive (Landrum et al. 2016) were excluded; (iii) variant with a frequency greater than 1% in gnomAD (Karczewski et al. 2020) were excluded.

Importantly, the TruSight^™ Oncology 500 measures homologous recombination deficiency (HRD). The HRD-related genes were as follows: ARID1A, ATM, ATRX, BAP1, BARD1, BLM, BRCA1, BRCA2, BRIP1, CHEK1, CHEK2, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCL, MRE11A, NBN, PALB2, PTEN, RAD50, RAD51, and RAD51B. Homologous recombination deficiency was diagnosed if there was at least one HR-related gene mutation.