RNA-based targeted next-generation sequencing

Targeted NGS-based testing from mRNA was also performed within the MGP lab at UPMC using the Oncomine Comprehensive Assay v3 (OCAv3) RNA primers (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s protocol. The OCAv3 RNA panel (Supplementary Table 1) evaluates 760 fusion genes that comprises intergenic and intragenic regions among 51 cancer-associated kinase genes. Briefly, 50ng mRNA is reverse transcribed into cDNA and subject to multiplex PCR to amplify the regions of interest. Amplicons were barcoded, ligated with specific adapters and purified. RNA library quantity and quality check were performed using the 4200 TapeStation (Agilent Technologies, Santa Clara, CA). The Ion Chef was used to prepare and enrich templates and enable testing via Ion Sphere Particles on a semiconductor chip. Massive parallel sequencing was carried out on an Ion S5 XL System according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA) and data was analyzed with the Torrent Suite Software v5.2.2 and an in-house bioinformatics program, Variant Explorer (UPMC) for RNA fusions and RNA expression. The limit of detection of the RNA assay was 1–5% tumor cells. More than 50 fusion-specific reads that cross the fusion breakpoint are required to make a positive fusion call.