Abstract 5632: Advancing transcriptome insights during RNA-Seq library construction of FFPE samples in a novel workflow

Abstract Formalin-fixed, paraffin-embedded (FFPE) tumor tissue has significant clinical benefits in oncology research and is considered vital for studying tumor gene expression through RNA sequencing (RNA-Seq). However, RNA extracted from FFPE samples tends to be severely degraded, making it suboptimal for constructing high-quality RNA-Seq libraries. The RNA-Seq workflow can further impair data quality, as traditional library preparation and pre-library ribodepletion workflows may result in the loss of valuable information due to rare transcripts being not captured or washed away. Moreover, small, degraded fragments are a challenging template for ligases, and the resulting adapter addition may be inadequate. Here we assessed a novel workflow employing a proprietary enzyme to overcome the limitations and biases of cDNA synthesis using reverse transcriptase (RT) and ligases. This innovative RNA-Seq library preparation approach, coupled with a post-library ribodepletion strategy, aims to capture a more complete transcriptome profile from RNA extracted from FFPE breast cancer samples, and can also be used for evaluation of gene regulation through intronic and intergenic bases. The study also compared two commercially available kits, RecoverAll and PureLink, for FFPE RNA extraction, evaluating quality metrics such as RNA integrity number (RIN), DV200, recovery of small RNAs during NGS library construction, and transcriptome coverage. A parallel fresh frozen RNA sample, extracted using a phenol-based reagent, served as a benchmark. Our findings demonstrate that the novel workflow yields a richer dataset, encompassing a wider range of transcriptomic elements, including both small and long RNAs from FFPE samples. Performing ribodepletion after the library preparation procedure led to increased detection of small RNAs compared to pre-library ribodepletion. The robust analytical performance of this streamlined library preparation protocol enables its suitability for RNA-Seq analysis of archived FFPE specimens, offering substantial utility for oncology research. Citation Format: Cissy Jiang, Elizabeth Jordan Dreskin, Angelica Olcott, Linda Lingelbach, Srikanth Perike. Advancing transcriptome insights during RNA-Seq library construction of FFPE samples in a novel workflow 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 5632.