TSS-Captur: a user-friendly pipeline for characterizing unclassified RNA transcripts.

RNA-seq and its 5'-enrichment methods for prokaryotes have enabled the precise identification of transcription start sites (TSSs), improving gene expression analysis. Computational methods are applied to these data to identify TSSs and classify them based on proximal annotated genes. While some TSSs cannot be classified at all (orphan TSSs), other TSSs are found on the reverse strand of known genes (antisense TSSs) but are not associated with the direct transcription of any known gene. Here, we introduce TSS-Captur, a novel pipeline, which uses computational approaches to characterize genomic regions starting from experimentally confirmed but unclassified TSSs. By analyzing TSS data, TSS-Captur characterizes unclassified signals, complementing prokaryotic genome annotation tools. TSS-Captur categorizes extracted transcripts as either messenger RNA for genes with coding potential or non-coding RNA (ncRNA) for non-translated genes. Additionally, it predicts the transcription termination site for each putative transcript. For ncRNA genes, the secondary structure is computed. Moreover, all putative promoter regions are analyzed to identify enriched motifs. An interactive report allows seamless data exploration. We validated TSS-Captur with a dataset and characterized unlabeled ncRNAs in . TSS-Captur is available both as a web-application and as a command-line tool.