A refined Saccharomyces cerevisiae reference transcriptome from Direct RNA Sequencing, with a reusable pipeline for UTR annotation updates

Abstract

Untranslated regions (UTRs) flanking the coding sequence govern mRNA translation, localisation, stability, and decay, making accurate UTR boundaries essential for quantitative RNA sequencing and the study of post-transcriptional control in Saccharomyces cerevisiae and beyond. Reference transcriptomes built from short-read sequencing have been invaluable to the yeast community, yet in a genome as gene-dense as that of S. cerevisiae, short reads frequently cannot be assigned to a single transcript of origin, leaving roughly one quarter of transcripts without a confidently defined UTR. Here we use Oxford Nanopore Direct RNA Sequencing (DRS), in which each full-length polyadenylated molecule is read end to end, to resolve this ambiguity and deliver two complementary resources. First, an updated, ready-to-use S. cerevisiae S288C reference: change-point segmentation of per-gene DRS coverage defined boundaries for 5,416 of the 6,695 annotated genes, and a merge-max rule retaining the longer UTR from each source ensures no gene loses existing annotation. The result adds previously absent UTRs to 927 (5') and 896 (3') genes and extends 29.4% of 5' and 26.1% of 3' boundaries among comparable genes. Second, the complete, documented pipeline so that any laboratory can rebuild or update a transcriptome from its own DRS data. Validation on two independent datasets shows improved mapping rates, reduced soft-clipping, and metagene profiles consistent with genuine transcript signal.