Optical Molecular Diagnostics and System Technology

Background: Genome annotation is an important step in deriving functional meaning from prokaryotic sequencing data, yet systematic evaluations guiding tool selection are lacking. We present the first large-scale investigation of four prominent opensource annotation tools (Prokka, Bakta, EggNOG-mapper, and PGAP) across 156,033 diverse genomes. This includes Escherichia coli strains for baseline performance, thousands of archaea and bacteria genomes, as well as frameshifted and metagenomeassembled genomes. Results: Bakta excels in annotating high-quality bacterial genomes, while PGAP was better for archaeal genomes and challenging bacterial assemblies, including metagenome-assembled, fragmented, or contaminated samples. For Gene Ontology annotation, PGAP consistently provides broader term coverage, whereas EggNOGmapper offers more terms per feature. Conclusions: Our findings highlight tool-specific strengths crucial for selecting optimal solutions based on genome quality, taxonomy, and origin (e.g. MAGs). This study provides an evidence-based guide for users and informs future tool development. Keywords: Bioinformatics, Genome annotation, Bacteria, Archaea, Benchmark, Computational biology, Metagenome-assembled genome, Functional annotation, Gene function prediction, Gene ontology