Trusight oncology 500 kit

The TruSight Oncology 500 Kit (TSO500, Illumina) was used for DNA library preparation and enrichment following the manufacturer’s protocol. DNA (80 ng) were sheared on a Covaris E220 ultrasonicator. DNA fragments were end-repaired, and adapters containing unique molecular identifiers (UMIs) were ligated to each fragment end. Fragments enriched by capture hybridization were analyzed by high-throughput sequencing on a NovaSeq 6000 instrument (Illumina). TSO500 alignment and variant calling were performed using the TSO500 bioinformatics pipeline v2.1.0. UMI-filtered total read counts were 103 M ± 19 M, median exon coverage was 1131 ± 253, median DNA-insert size was 136 ± 14, and % aligned reads were 98.9 ± 1.0. Sources of population frequencies that were used for auto-classification of benign variation include gnomAD (RRID:SCR_014964) and ExAC (RRID:SCR_004068). We retrieved annotations of oncogenic effects of identified variants from the OncoKB precision oncology knowledge database (RRID:SCR_014782) and assessed known activating mutations in oncogenes and inactivating mutations in tumor suppressors (Tier 1 and Tier 2). For the few cases in which IHC was inconclusive, we investigated both known mutations and mutations with unknown and unclear effects (Tier 3). OncoPrint function from ComplexHeatmap v2.6.2^{45 (link)} (RRID:SCR_017270) was used for visualization.

DNA was extracted from formalin-fixed and paraffin-embedded (FFPE) tumor tissue and non-lesional adjacent liver tissue using Maxwell CSC instrument (Promega, Madison, WI, USA) with the Maxwell RSC DNA FFPE kit (Promega, Madison, WI, USA) according to the manufacturer’s protocols; DNA concentrations were measured on a Qubit 2.0 fluorometer (Thermofisher Scientific, Waltham, MA, USA) using the Qubit dsDNA High Sensitivity Assay Kit.
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 550 (Illumina Inc., San Diego, CA, USA). The quality of the NextSeq 550 (Illumina) sequencing runs was assessed with the Illumina Sequencing Analysis Viewer (Illumina). NGS data were analyzed with Illumina TruSight Oncology 500 Local App v2.1 [11 ], and variant report files were uploaded into the Pierian Clinical Genomics Workspace cloud (Pierian DX software CGW_V6.21.1).

40 ng DNA were quantified with the Qubit dsDNA HS Assay (Thermo Fisher Scientific) on the Qubit 2.0 Fluorometer (Thermo Fisher Scientific) and sheared on the Covaris E220 Focused-ultrasonicator (Woburn, MA, USA) using the 8 microTUBE–50 Strip AFA Fiber V2 following manufacturer’s instructions. The treatment time was optimized for FFPE material. The treatment settings were the following: Peak Incident Power (W): 75; Duty Factor: 15%; Cycles per Burst: 500; Treatment Time (s): 360; Temperature (°C): 7; Water Level: 6. For DNA library preparation and enrichment the TruSight Oncology 500 Kit (Illumina) was used following manufacturer’s instructions. Post-enriched libraries were quantified, pooled and sequenced on a NextSeq 500 (Illumina Inc., San Diego, CA, USA).
Quality of the NextSeq 500 (Illumina) sequencing runs were assessed with the Illumina Sequencing Analysis Viewer (Illumina). Sequencing data was analyzed with the TruSight Oncology 500 Local App Version 1.3.0.39 (Illumina).

The presence of NTRK fusions in patients receiving TRK inhibitor therapy was identified by commercially available oncology genomic profiling assays, including Archer Fusionplex Lung or OncologyResearch panels (IDT, ArcherDX), Oncomine Comprehensive Assay v3 (Thermo Fisher), Oncomine Focus Assay (Thermo Fisher), TruSight Tumor 170 kit (Illumina) and TruSight Oncology 500 kit (Illumina) from primary or metastatic tissue, according to the procedures established by each laboratory (Kirchner et al. 2020 (link); Pfarr et al. 2020 (link)).

Libraries for mutational profiling were prepared, starting from 40 ng of DNA as input material, with TruSight™ Oncology500 kit (Cat. 20028213, Illumina, San Diego, California, USA) according to manufacturer’s protocol. These consist in a targeted-capture of 523 cancer-relevant genes. The libraries were sequenced on NextSeq 500 (Illumina) in 2 × 150 bp or 2 × 75 bp in paired end mode. Sequencing data were analyzed using the TruSight Oncology 500 Local App Version 2.2 (Illumina) to identify variants, gene amplifications, TMB and MSI. The genomic coordinates of all the identified variants were subsequently converted to hg38 using LiftOver [31 (link)].
Variants were annotated with Annovar [32 (link)]. Those with coverage depth lower than 100 and variants occurring with a frequency higher than 5% in 1000G or GnomAD were discarded. The sequence variants annotated as “benign” or “likely benign” in ClinVar database were also filtered out. Furthermore, variants classified as synonymous or in intergenic positions were discarded. Oncoplots were generated using Maftools [33 (link)] on R (v4.0.2). Functional analysis was performed using Ingenuity Pathway Analysis (IPA, Qiagen, Hilden, Germany) and only the pathways with more than 1.3 in –log of the adjusted p-value were considered.