A refined Saccharomyces cerevisiae reference transcriptome from direct RNA sequencing, with a reusable pipeline for UTR annotation updates.

Untranslated regions (UTRs) flanking the coding sequence govern mRNA translation, localization, and decay, making accurate UTR boundaries essential for quantitative RNA sequencing and the study of post-transcriptional control. Reference transcriptomes built from short-read sequencing have been invaluable to the yeast community, yet in a genome as gene-dense as that of Saccharomyces cerevisiae, short reads cannot be assigned to a single transcript of origin, leaving roughly one quarter of transcripts without a defined UTR. Here, we use direct RNA sequencing (DRS), in which native polyadenylated molecules are sequenced without fragmentation or amplification, to reduce this ambiguity and deliver two complementary resources. First, an updated S. cerevisiae S288C reference: change-point segmentation of per-gene DRS coverage defined boundaries for 5682 of the 6607 genes in the backbone model, 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 1213 (5') and 1220 (3') of the genes that the earlier short-read annotation left unannotated, and extends 28.9% of 5' and 19.4% of 3' boundaries among comparable genes. Second, the complete, documented pipeline so that the transcriptome can be rebuilt from any DRS data. Validation on two independent datasets shows improved mapping rates, reduced soft-clipping, and metagene profiles consistent with genuine transcript signal.