Development Of Customizable Targeted Rna Fusion Panels Using A Novel Automated High-Multiplexing Primer Design Strategy

Introduction Gene fusions play an important role in oncogenesis and the progression of cancer. As important biomarkers, sensitive identification of gene fusions is critical to future oncology research. Next generation sequencing with Ion Ampliseq targeted enrichment enables simple, accurate and specific detection of relevant fusion isoforms. Here we introduce a novel automatic high-multiplexing primer design strategy that has the flexibility to develop customized Ampliseq fusion panels for any combination of fusion isoforms, scaling to panels that can detect thousands of isoforms in a single primer pool, which increases the sensitivity of fusion detection while decreasing the sample input required to as low as 10 ng. Methods The automated primer design pipeline takes a Gene-Transcript-Exon (GTE) file as input. Each record in the GTE file represents a unique RNA fusion isoform to establish an easy-parsing format for the pipeline. The pipeline locates the fusion breakpoint position, extracts gene sequences of every candidate fusion target and builds the fusion reference. Candidate amplicons are generated against the fusion reference. According to the design requirements of pool number and the conflicts among primer pairs, the pipeline performs pooling to minimize primer interactions. Finally, the pipeline generates an optimal set of amplicons strategically targeted for fusion junctions. The output files are used for downstream analysis with a fully automated analysis pipeline. Results The pipeline has been used extensively to develop high performing multiplex RNA fusion panels. The pipeline generates 175-base amplicons for use on formalin-fixed, paraffin-embedded (FFPE) samples or 120-base amplicons for use on cfRNA from blood samples. A single panel can include thousands of known fusion variants. This pipeline has been used to design the fusion assays contained in Oncomine Focus and Comprehensive assays, Oncomine Precision Assay, and others. Oncomine Comprehensive Assay v3 fusion panel was tested using the Ion GeneStudio S5 Sequencer; for example, testing on SeraCare fusion control confirms that all 14 fusion isoforms were detected with 100% accuracy. Testing on FFPE samples with known positive fusions confirms that the expected fusions including NTRK1, ERG, ETV1 and MET driver genes were also detected with 100% accuracy. Conclusions In summary, we have developed an automatic pipeline that can generate robust, comprehensive customized multiplex RNA fusion assays for targeted next-generation sequencing. For research use only. Not for use in diagnostic procedures. Citation Format: NA LI, Antonio Martinez-Alcantara, Aren Ewing, Rajesh Gottimukkala, Fiona Hyland, Seth Sadis. Development of customizable targeted RNA fusion panels using a novel automated high-multiplexing primer design strategy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5472.