Abstract Motivation Ribosome profiling (Ribo-seq) is a powerful approach based on ribosome-protected RNA fragments to explore the translatome of a cell, and is especially useful for the detection of small proteins (&lt;=70 amino acids) that are recalcitrant to biochemical and in silico approaches. While pipelines are available to analyze Ribo-seq data, none are designed explicitly for the analysis of Ribo-seq data from prokaryotes, nor are they focused on the discovery of unannotated open reading frames (ORFs) in bacteria. Results We present HRIBO (High-throughput annotation by Ribo-seq), a workflow to enable reproducible and high-throughput analysis of bacterial Ribo-seq data. The workflow performs all required pre-processing and quality control steps. Importantly, HRIBO outputs annotation-independent ORF predictions based on two complementary bacteria-focused tools, and integrates them with additional features. This facilitates the rapid discovery of novel ORFs and their prioritization for functional characterization. Availability HRIBO is a free and open source project available under the GPL-3 license at: https://github.com/RickGelhausen/HRIBO

HRIBO - High-throughput analysis of bacterial ribosome profiling data