Large-scale Evaluation of Prokaryotic Annotation Tools Across Thousands of Species

Large-scale Evaluation of Prokaryotic Annotation Tools Across Thousands of Species | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Analysis Large-scale Evaluation of Prokaryotic Annotation Tools Across Thousands of Species Mateusz Jundzill, Martin Hölzer, Serghei Mangul, Mike Marquet, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7317585/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Genome annotation is fundamental to prokaryotic genome sequencing, yet systematic evaluations guiding tool selection are lacking. We present the first large-scale investigation of four prominent open-source annotation tools (Prokka, Bakta, EggNOG-mapper, and PGAP) across 156,033 diverse genomes, including Escherichia coli strains for baseline performance, thousands of archaea and bacteria, frameshifted, and metagenome-assembled genomes. Bakta excelled in annotating high-quality bacterial genomes, while PGAP was better for archaeal and challenging genomes, including bacterial metagenome-assembled, fragmented, or contaminated samples. For biological functional Gene Ontology annotation, PGAP provided broader term coverage, whereas EggNOG-mapper offered more terms per feature. 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. Biological sciences/Computational biology and bioinformatics/Sequence annotation Biological sciences/Microbiology Biological sciences/Computational biology and bioinformatics/Standards Biological sciences/Molecular biology Full Text Additional Declarations There is NO Competing Interest. Supplementary Files Supplementaryrawdata.xlsx Supplementary raw data annotationsupplement.pdf Main Supplement Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7317585","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Analysis","associatedPublications":[],"authors":[{"id":497091904,"identity":"72a726e0-30a0-4aa0-9cc6-eb82fef64f7d","order_by":0,"name":"Mateusz 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