Targeted RNA Sequencing of Primary Tumors

In three cases, targeted RNA sequencing was performed on samples of primary tumors. Total RNA was extracted from formalin-fixed paraffin-embedded tissue scrolls (3–4 per case) using the ExpressArt FFPE Clear RNA Ready kit (Amsbio, Cambridge, MA); it was assessed using the RNA 6000 Nano Bioanalyzer Kit (Agilent, Mississauga, ON) and quantitated using the Qubit RNA HS Assay Kit (ThermoFisher Scientific, Mississauga, ON). An input of 20–100 ng total RNA and the TruSight RNA Fusion Panel were used to prepare the RNA-seq libraries (Illumina, San Diego, CA), following manufacturer’s instructions and as previously described.^{7 (link),8 (link)} Sequencing of each sample was performed with 76 base-pair paired-end reads on an Illumina MiSeq at eight samples per flow cell (~3 million reads per sample). The results were then analyzed using the STAR and BOWTIE2 aligners, and Manta and JAFFA fusion callers, respectively.^{9 (link),10 (link)}