Seraseq Fusion RNA-seq

Seraseq Fusion RNA Mix was utilized as a gold standard reference material to test the ensemble fusion detection pipeline. This product was engineered in the GM24385 cell line and contains 14 synthetic gene fusion events. For undiluted (neat) samples, RNA-Seq libraries were prepared utilizing Seraseq Fusion RNA v2 Dilution Panel RNA (SeraCare). RNA-seq libraries were prepared for diluted samples and negative controls using Seraseq Fusion RNA v3 (SeraCare), which is roughly equivalent to a 1:25 dilution of the v2 product. Concentration of individual fusions were determined by a custom fluorescent probe set (based on TaqMan probe design) evaluated by droplet digital PCR. To obtain dilutions of the Seraseq Fusion RNA v3 product (initially a 1:25 dilution), Seraseq Fusion RNA v3 was mixed with control total RNA (GM24285, Coriell) for final dilutions of 1:25, 1:50, 1:250, 1:500, 1:2500, and 0. For neat and diluted samples, 500ng input RNA was ribodepleted using the NEBNext Human/Mouse/Rat rRNA Depletion kit and libraries were prepared following the NEBNext Ultra II Directional RNA-Seq protocol (New England BioLabs). 2x151 paired ends reads were sequenced using the HiSeq4000 (Illumina). An average of 148.7 million reads were obtained per Seraseq sample, with a minimum of 85.8 million reads and a maximum of 227.0 million reads.