Abstract 321: Optimized RNA Seq library preparation for fusion calling from FFPE samples

Abstract Gene fusions due to chromosomal rearrangement, duplication, or deletion can be important drivers of cancer. Detection of gene fusions plays a valuable role in selecting targeted therapies. A widely used method for detecting fusion transcripts is targeted RNA sequencing, which focuses sequencing reads on known fusions but does not allow for the detection of novel fusions. Whole transcriptome analysis (WTA), where the entire transcriptome is interrogated, offers an opportunity to detect both known and novel fusions. Herein we examine the effect of sample quality, input mass, increased reverse transcriptase (RT) and insert size on fusion calling. Libraries are prepared using the Watchmaker RNA Library Preparation Kit with or without Polaris Depletion and, for target enrichment, the Twist Exome 2. 0 panel prior to paired end 2 × 150 Illumina sequencing. First, control samples, specifically Seraseq Fusion RNA v4 high quality and FFPE samples, are used to determine if any of the parameters interrogated increase the number of breakpoint spanning reads both in a WTA and targeted sequencing context. Once established, the optimized workflow is applied to real FFPE samples. Targeted sequencing results in a higher number of breakpoint spanning reads, compared to WTA, when controlling for sequencing depth, resulting in a higher number of reads supporting each fusion call. Longer insert sizes, achieved by modified SPRI ratios, maintain library complexity and may enhance fusion calling. Taken together, these data demonstrate an optimized protocol for fusion calling from FFPE and demonstrate the utility of both WTA and targeted sequencing for fusion detection. Citation Format: Giulia Corbet, Josh Haimes, Travis Sanders, Martin Ranik, Thomas Harrison, Eduard Casas, Kailee Reed, Doug Wendel, Kristina Giorda, Ariele Hanek, Jen Pavlica, Brian Kudlow. Optimized RNA Seq library preparation for fusion calling from FFPE samples abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 321.