biostudies-literatureUnknown39(7)Syberg-Olsen MJProkaryotic genomes are usually densely packed with intact and functional genes. However, in certain contexts, such as after recent ecological shifts or extreme population bottlenecks, broken and nonfunctional gene fragments can quickly accumulate and form a substantial fraction of the genome. Identification of these broken genes, called pseudogenes, is a critical step for understanding the evolutionary forces acting upon, and the functional potential encoded within, prokaryotic genomes. Here, we present Pseudofinder, an open-source software dedicated to pseudogene identification and analysis in bacterial and archaeal genomes. We demonstrate that Pseudofinder's multi-pronged, reference-based approach can detect a wide variety of pseudogenes, including those that are highly degraded and typMolecular biology and evolutionmsac153https://www.ebi.ac.uk/biostudies/studies/S-EPMC9336565biostudies-literaturePseudofinder: Detection of Pseudogenes in Prokaryotic Genomes.PMC9336565Garber AISyberg-Olsen MJHusnik FKeeling PJMcCutcheon JPfalsePseudofinder: Detection of Pseudogenes in Prokaryotic Genomes.Prokaryotic genomes are usually densely packed with intact and functional genes. However, in certain contexts, such as after recent ecological shifts or extreme population bottlenecks, broken and nonfunctional gene fragments can quickly accumulate and form a substantial fraction of the genome. Identification of these broken genes, called pseudogenes, is a critical step for understanding the evolutionary forces acting upon, and the functional potential encoded within, prokaryotic genomes. Here, we present Pseudofinder, an open-source software dedicated to pseudogene identification and analysis in bacterial and archaeal genomes. We demonstrate that Pseudofinder's multi-pronged, reference-based approach can detect a wide variety of pseudogenes, including those that are highly degraded and typ2022-01-01T00:00:00Z2022 Jul2025-04-05T08:56:27.076Z2025-04-05T08:56:27.076ZS-EPMC93365653580156210.1093/molbev/msac153