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We present a high-throughput technique \u2013 Quantitative Mutational Scan sequencing (QMS-seq) \u2013 that enables quantitative comparison of which genes are under antibiotic selection and captures how genetic background influences resistance evolution. We compare four\n                    <jats:italic>E. coli<\/jats:italic>\n                    strains exposed to ciprofloxacin, cycloserine, or nitrofurantoin and identify 812 resistance mutations, many in genes and regulatory regions not previously associated with resistance. We find that multi-drug and antibiotic-specific resistance are acquired through categorically different types of mutations, and that minor genotypic differences significantly influence evolutionary routes to resistance. By quantifying mutation frequency with single base pair resolution, QMS-seq informs about the underlying mechanisms of resistance and identifies mutational hotspots within genes. Our method provides a way to rapidly screen for resistance mutations while assessing the impact of multiple confounding factors.\n                  <\/jats:p>","DOI":"10.1038\/s41467-025-56050-2","type":"journal-article","created":{"date-parts":[[2025,1,17]],"date-time":"2025-01-17T10:59:35Z","timestamp":1737111575000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":22,"title":["High-throughput method characterizes hundreds of previously unknown antibiotic resistance mutations"],"prefix":"10.1038","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-9511-2020","authenticated-orcid":false,"given":"Matthew J.","family":"Jago","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7131-2560","authenticated-orcid":false,"given":"Jake K.","family":"Soley","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6465-3818","authenticated-orcid":false,"given":"Stepan","family":"Denisov","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Calum J.","family":"Walsh","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4990-8816","authenticated-orcid":false,"given":"Danna R.","family":"Gifford","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0237-1473","authenticated-orcid":false,"given":"Benjamin P.","family":"Howden","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7847-3594","authenticated-orcid":false,"given":"Mato","family":"Lagator","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,1,17]]},"reference":[{"key":"56050_CR1","doi-asserted-by":"publisher","first-page":"629","DOI":"10.1016\/S0140-6736(21)02724-0","volume":"399","author":"CJL Murray","year":"2022","unstructured":"Murray, C. 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