Poster Presentation 45th Lorne Genome Conference 2024

Falling short in long-read transcriptomics: Is “full-length” really full-length? (#127)

James Lancaster 1 , Quentin Gouil 1 , Matthew Ritchie 1
  1. WEHI, Parkville, VIC, Australia

Short-read RNA sequencing excels at quantifying gene-level expression thanks to streamlined library preparation workflows and the possibility to sequence at high depth (billions of reads on the NovaSeq platform). However, defining and quantifying isoforms remains a challenge with short-read sequencing. On the other hand, long-read sequencing platforms such as Oxford Nanopore (ONT) and PacBio are unveiling the huge complexity of transcriptomes. These platforms enable the sequencing of complete transcripts in a single read, though long transcripts (>3kb) remain challenging and are rarely sequenced in full-length by the Nanopore PCR-cDNA protocol. In this study we evaluated the potential of the new Induro reverse transcriptase to improve full-length sequencing of long transcripts. We benchmarked multiple experimental conditions on synthetic transcripts (Lexogen SIRV long module), and improved analysis tools for transcript coverage calculation and visualisation. Despite minimal improvements in sequenced read length compared to Maxima H minus, our findings underscore the persisting challenges in long-read transcriptomics.