Simon Haile ^1* Richard D Corbett ¹ Kieran O’Neill ¹ Jing Xu ¹ Duane E Smailus ¹ Pawan K Pandoh ¹ Anthony Bayega ¹ Miruna Bala ¹ Eric Chuah ¹ Robin Coope ¹ Richard A Moore ¹ Karen L Mungall ¹ Yongjun Zhao ¹ Yussanne Ma ¹ Marco Marra ^1,2 Steven Jones ^1,2 Andrew J Mungall ¹

• ¹ Canada's Michael Smith Genome Sciences Centre, Vancouver, Canada
• ² University of British Columbia, Vancouver, British Columbia, Canada

The advent of long-read (LR) sequencing technologies has provided a direct opportunity to determine the structure of transcripts with potential for end-to-end sequencing of full-length RNAs. LR methods that have been described to date include commercial offerings from Oxford Nanopore Technologies (ONT) and Pacific Biosciences. These kits are based on selection of polyadenylated (polyA+) RNAs and/or oligo-dT priming of reverse transcription. Thus, these approaches don’t allow comprehensive interrogation of the transcriptome due to their exclusion of non-polyadenylated (polyA-) RNAs. In addition, polyA+ specificity also results in 3’-biased measurements of PolyA+ RNAs especially when the RNA input is partially degraded. To address these limitations of current LR protocols, we modified rRNA depletion protocols that have been used in short-read sequencing: one approach representing a ligation-based method and the other a template-switch cDNA synthesis-based method to append ONT-specific adaptor sequences and by removing any deliberate fragmentation/shearing of RNA/cDNA. Here, we present comparisons with poly+ RNA-specific versions of the two approaches including the ONT PCR-cDNA Barcoding kit. The rRNA depletion protocols displayed higher proportions (30-50%) of intronic content compared to that of the polyA-specific protocols (5-8%). In addition, the rRNA depletion protocols enabled ~20-50% higher detection of expressed genes. Other metrics that were favourable to the rRNA depletion protocols include better coverage of long transcripts, and higher accuracy and reproducibility of expression measurements. Overall, these results indicate that the rRNA depletion-based protocols described here allow the comprehensive characterization of polyadenylated and non-polyadenylated RNAs. While the resulting reads are long enough to help decipher transcript structures, future endeavors are warranted to improve the proportion of individual reads representing end-to-end spanning of transcripts.