Application of the 3’ mRNA-Seq using unique molecular identifiers in highly degraded RNA derived from formalin-fixed, paraffin-embedded tissue

Background: Archival formalin-fixed, paraffin-embedded (FFPE) tissue samples with clinical and histological data are a singularly valuable resource for developing new molecular biomarkers. However, transcriptome analysis remains challenging with standard mRNA-seq methods as FFPE derived-RNA samples are often highly modified and fragmented. The recently developed 3’ mRNA-seq method sequences the 3’ region of mRNA using unique molecular identifiers (UMI), thus generating gene expression data free from PCR amplification bias. In this study, we evaluated the performance of the 3’ mRNA-Seq using Lexogene QuantSeq 3’ mRNA-Seq Library Prep Kit FWD with UMI, comparing with TruSeq stranded mRNA-Seq and RNA Exome Capture. The fresh-frozen (FF) and FFPE tissues yielded nucleotide sizes range from 13% to >70% of DV200 values; input amounts ranged from 1ng to 100ng for validation. Results: The total mapped reads of the 3’ mRNA-Seq to the reference genome ranged from 99% to74% across all the samples. After PCR bias correction, total mapped reads ranged from 3% to 56%. The 3’ mRNA-Seq data showed highly reproducible data in the UHR (R>0.94) at different input amounts from 1ng to 100ng, and FF and FFPE paired samples (R=0.93) at 10 ng. Severely degraded FFPE RNA with 13% to 30% of DV200 value showed good concordance (R>0.90) with 100ng input. A moderate correlation was observed when directly comparing 3’ mRNA-Seq data with TruSeq stranded mRNA-Seq (R=0.78) and RNA Exome Capture data (R>0.67). Conclusion: In this study, 3’ mRNA-Seq with PCR bias correction using UMI is a suitable method for gene quantification in both FF and FFPE RNAs. The 3’ mRNA-Seq may be applied to severely degraded RNA from FFPE tissues and generate high-quality sequencing data. The 3’ mRNA-Seq using Lexogene QuantSeq 3’ mRNA-Seq Library Prep Kit FWD with UMI is compared with TruSeq stranded mRNA-Seq and RNA Exome Capture kits. The fresh-frozen (FF) and FFPE tissues yielded nucleotide sizes range from 13% to >70% of DV200 values; input amounts ranged from 1ng to 100ng for validation.