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Using Mycobacterium tuberculosis, a clonally evolving bacterium with a small accessory genome, as a model system, we systematically evaluated sources of variability in pan-genome estimates. Our analysis revealed that differences in assembly type (short-read versus hybrid), annotation pipeline, and pan-genome software, significantly impact predictions of core and accessory genome size. Extending our analysis to two additional bacterial species, Escherichia coli and Staphylococcus aureus, we observed consistent tool-dependent biases but species-specific patterns in pan-genome variability. Our findings highlight the importance of integrating nucleotide- and protein-level analyses to improve the reliability and reproducibility of pan-genome studies across diverse bacterial populations.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Availability and implementation<\/jats:title>\n                    <jats:p>Panqc is freely available under an MIT license at https:\/\/github.com\/maxgmarin\/panqc.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1093\/bioinformatics\/btaf219","type":"journal-article","created":{"date-parts":[[2025,5,8]],"date-time":"2025-05-08T07:46:47Z","timestamp":1746690407000},"source":"Crossref","is-referenced-by-count":15,"title":["Pitfalls of bacterial pan-genome analysis approaches: a case study of\n                    <i>Mycobacterium tuberculosis<\/i>\n                    and two less clonal bacterial species"],"prefix":"10.1093","volume":"41","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9108-3328","authenticated-orcid":false,"given":"Maximillian G","family":"Marin","sequence":"first","affiliation":[{"name":"Department of Biomedical Informatics, Harvard Medical School , Boston, MA 02115,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Natalia","family":"Quinones-Olvera","sequence":"additional","affiliation":[{"name":"Department of Biomedical Informatics, Harvard Medical School , Boston, MA 02115,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christoph","family":"Wippel","sequence":"additional","affiliation":[{"name":"Department of Biomedical Informatics, Harvard Medical School , Boston, MA 02115,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mahboobeh","family":"Behruznia","sequence":"additional","affiliation":[{"name":"Department of Biosciences, Nottingham Trent University , Nottingham, NG1 4FQ,","place":["United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Brendan M","family":"Jeffrey","sequence":"additional","affiliation":[{"name":"Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health , Bethesda, 20892,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael","family":"Harris","sequence":"additional","affiliation":[{"name":"Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health , Bethesda, 20892,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Brendon C","family":"Mann","sequence":"additional","affiliation":[{"name":"Centre of Excellence for Biomedical Tuberculosis Research, South African Medical Research Council Centre for Tuberculosis 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