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We introduce Seqwin, an open-source framework designed to automate microbial genome signature discovery. Tens of thousands of microbial genomes are now available for a single species, limiting the application of existing manual and automated approaches for identifying signatures. Modern approaches that are capable of leveraging all available microbial genomes will ensure sensitive and accurate DNA signature identification and enable robust pathogen detection for clinical, environmental, and public health applications.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>Seqwin builds weighted pan-genome minimizer graphs and uses a traversal algorithm to identify signature sequences that occur frequently in target genomes but remain rare in non-targets. Unlike earlier tools that depend on strict presence or absence of sequences, Seqwin accommodates natural sequence variation and scales to very large genome collections. When applied to genomes from C. difficile, M. tuberculosis, and S. enterica, Seqwin recovered more high-quality signatures than alternative methods with lower computational burden. Seqwin\u2019s analysis of nearly 15\u2009000 S. enterica genomes yielded over 200 candidate signatures in three minutes. Seqwin provides an open-source solution for the long-standing need for scalable microbial signature discovery and diagnostic assay design.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Availability and Implementation<\/jats:title>\n                    <jats:p>Seqwin is available on GitHub (https:\/\/github.com\/treangenlab\/Seqwin) and can be installed via Bioconda (https:\/\/bioconda.github.io\/recipes\/seqwin\/README.html). Benchmarking datasets, outputs, and scripts are available on Zenodo (https:\/\/doi.org\/10.5281\/zenodo.19874011).<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1093\/bioinformatics\/btag261","type":"journal-article","created":{"date-parts":[[2026,7,7]],"date-time":"2026-07-07T16:32:14Z","timestamp":1783441934000},"source":"Crossref","is-referenced-by-count":0,"title":["Seqwin: ultrafast identification of signature sequences in microbial genomes"],"prefix":"10.1093","volume":"42","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7009-6958","authenticated-orcid":false,"given":"Michael X","family":"Wang","sequence":"first","affiliation":[{"name":"Department of Computer Science, Rice University , Houston, TX 77005,","place":["United States"]},{"name":"Department of Civil and Environmental Engineering, Rice University , Houston, TX 77005,","place":["United 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