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

Area of Science:

• Genomics
• Computational Biology
• Molecular Biology

Background:

• RNA sequencing (RNA-seq) with 5'-enrichment methods precisely identifies transcription start sites (TSSs) in prokaryotes, enhancing gene expression analysis.
• Computational tools classify TSSs based on proximity to annotated genes, but unclassified (orphan) and antisense TSSs remain challenging.
• Characterizing these unclassified TSSs is crucial for comprehensive prokaryotic genome annotation.

Purpose of the Study:

• To introduce TSS-Captur, a novel computational pipeline for characterizing unclassified transcription start sites (TSSs) in prokaryotes.
• To complement existing prokaryotic genome annotation tools by analyzing unclassified TSS data.
• To categorize transcripts, predict termination sites, analyze secondary structures of ncRNAs, and identify promoter motifs.

Main Methods:

• TSS-Captur utilizes computational approaches to analyze experimentally confirmed but unclassified TSSs.
• It categorizes transcripts into messenger RNA (mRNA) based on coding potential or non-coding RNA (ncRNA).
• The pipeline predicts transcription termination sites, computes ncRNA secondary structures, and analyzes promoter regions for enriched motifs.

Main Results:

• TSS-Captur successfully characterized unclassified genomic regions originating from experimentally confirmed TSSs.
• The pipeline effectively categorized transcripts as mRNA or ncRNA and predicted termination sites.
• Validation on *Campylobacter jejuni* and characterization of unlabeled ncRNAs in *Streptomyces coelicolor* demonstrated its utility.

Conclusions:

• TSS-Captur provides a valuable tool for characterizing unclassified TSSs in prokaryotes, thereby enhancing genome annotation.
• It facilitates the discovery and classification of novel coding and non-coding RNA genes.
• The pipeline offers an interactive report and is available as both a web application and a command-line tool.