SureSelect XT HS Custom Panel (Agilent)

Custom capture probes were designed using SureDesign (Agilent) targeting known fusion breakpoints and novel fusions in ALK, ROS1, RET, BRAF, MET, FGFR1-3, NTRK1-3 (Additional file 2: Table S2). 200 ng of DNA was sheared on the Covaris E220 Focused-ultrasonicator (Covaris, Woburn, MA, USA) to a fragment size of 150 bp 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): 175; Duty Factor: 10%; Cycles per Burst: 200; Treatment Time (s): 200; Temperature (°C): 7; Water Level: 6.

For library preparation the SureSelect XT HS Reagent Kit (Agilent) was used according to manufacturer’s instructions. In brief, pre-enriched adapter-ligated libraries were prepared. Subsequently, custom capture probes were hybridized to target sequences to allow for sequence enrichment using streptavidin beads. Post-enriched libraries were quantified using the Qubit dsDNA Assay Kit (Thermo Fisher Scientific) and library quality was assessed using the Fragment Analyzer (Agilent). Libraries were pooled to equimolar concentrations and sequenced on a NextSeq 500 (Illumina). Sequencing data were analyzed with the SureCall Software v4.0.1.46 and v4.1.1.5 (Agilent). The correlation coefficient (R) was calculated with the following formula: Σ [(X − X_m) * (Y − Y_m)] / √ [Σ (X − X_m)² * Σ (Y − Y_m)²]. The coefficient of determination (R²) is the squre of the correlation coefficient. A fusion was classified as present with among others the following setting: Minimum number of reads per translocation > 5. The time for analysis heavily relied on the used hardware. On our local hardware one sample took around 1.5–2 h.