Prokanota: a modular, configuration-driven workflow for prokaryotic genome annotation
Background Prokaryotic genome annotation is central to comparative genomics, functional interpretation, and hypothesis generation. Established tools such as Prokka and Bakta provide streamlined annotation workflows, but predefined database choices and hierarchical annotation strategies can limit flexibility, especially for non-model organisms or specialized tasks. In contrast, custom pipelines are more adaptable but usually require substantial bioinformatic expertise to build and maintain. Methods We developed Prokanota (PROKAryotic ANnOTAtion), a modular workflow combining genomic feature prediction, including coding sequences, RNA genes, and CRISPR loci, with configurable functional annotation and standardized output generation. Prokanota is implemented as a Snakemake-based command-line tool and uses configuration files to define inputs, outputs, and annotation databases. It integrates pyrodigal for coding sequence prediction, pybarrnap for ribosomal RNA prediction, tRNAscan-SE for transfer RNA prediction, and diced for CRISPR loci prediction. Functional annotation is based on user-defined database modules and currently supports pyhmmer, RPS-BLAST, DIAMOND, and MMseqs2 search backends. Results Prokanota generates input-derived deterministic feature identifiers and synchronized GFF, TSV, GenBank, nucleotide FASTA, and protein FASTA outputs. Its modular database architecture enables users to combine general-purpose and specialist resources while retaining database-specific top-hit information. In an evaluation of eight bacterial and eight archaeal reference genomes, combining databases increased the proportion of CDS classified as annotated, particularly in Archaea. Combining CDD, KOfam, and arCOG databases yielded the highest annotation coverage for all tested archaeal genomes, with 81–90% of predicted CDS classified as annotated, rendering Prokanota especially useful for the annotation of archaeal genomes. Conclusions Prokanota fills a practical gap between fixed, standardized annotation tools and fully custom expert-built workflows. It provides an accessible and reproducible annotation backbone while allowing project-specific databases and evidence layers to be incorporated without modifying workflow code. The software is freely available under the Boost Software License 1 at https://github.com/richardstoeckl/prokanota.