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To address these challenges, we developed\n                  <jats:italic>bronko<\/jats:italic>\n                  , an ultrafast alignment-free framework for detecting viral variation directly from sequencing data. The novel computational approach implemented in\n                  <jats:italic>bronko<\/jats:italic>\n                  allows scaling to massive viral sequencing datasets and has three key components: i) a locality-sensitive bucketing function to rapidly identify single-nucleotide polymorphisms (SNPs) relative to reference(s), ii) a direct k-mer count psuedo-mapping approach that approximates a pileup without alignment, and iii) a streaming-based sliding window outlier test to estimate baseline noise across the genome and precisely differentiate real minor variants from noise. Together, these components yield near-linear computational complexity with respect to sequencing depth, enabling bronko to process thousands of viral samples rapidly on modest hardware. Our results are threefold: 1) On simulated amplicon sequencing,\n                  <jats:italic>bronko<\/jats:italic>\n                  recovers variants with higher precision and comparable recall to existing tools while running up to one to three orders of magnitude faster; 2)\n                  <jats:italic>bronko<\/jats:italic>\n                  generates sequence alignments directly from sequencing data, with SNP content similar to that of whole-genome alignment while also running in a fraction of the time, and 3) applying\n                  <jats:italic>bronko<\/jats:italic>\n                  to longitudinal sequencing data from chronically infected SARS-CoV-2 patients revealed consistent patterns of intrahost diversification and adaptive mutations over time. Altogether, these results demonstrate\n                  <jats:italic>bronko<\/jats:italic>\n                  's potential as a scalable tool for large-scale viral genomic analyses, overcoming longstanding computational barriers for intrahost and interhost characterization of viral variation.\n                <\/jats:p>","DOI":"10.64898\/2025.12.01.691650","type":"posted-content","created":{"date-parts":[[2025,12,8]],"date-time":"2025-12-08T14:38:39Z","timestamp":1765204719000},"source":"Crossref","is-referenced-by-count":1,"title":["bronko: ultrafast, alignment-free detection of viral genome variation"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-8355-0926","authenticated-orcid":false,"given":"Ryan D","family":"Doughty","sequence":"first","affiliation":[{"name":"Rice University;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1168-1617","authenticated-orcid":false,"given":"Michael J","family":"Tisza","sequence":"additional","affiliation":[{"name":"Baylor College of Medicine"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3760-564X","authenticated-orcid":false,"given":"Todd J","family":"Treangen","sequence":"additional","affiliation":[{"name":"Rice University;"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"issue":"3","key":"2025120800592408000_2025.12.01.691650v1.1","doi-asserted-by":"crossref","first-page":"1431","DOI":"10.1042\/BST20231322","article-title":"Viral genome sequencing methods: benefits and pitfalls of current approaches","volume":"52","year":"2024","journal-title":"Biochemical Society Transactions"},{"key":"2025120800592408000_2025.12.01.691650v1.2","doi-asserted-by":"publisher","DOI":"10.1038\/nrmicro.2016.182"},{"issue":"8","key":"2025120800592408000_2025.12.01.691650v1.3","doi-asserted-by":"crossref","first-page":"1229","DOI":"10.1038\/s12276-021-00658-z","article-title":"Mutational spectrum of SARS-CoV-2 during the global pandemic","volume":"53","year":"2021","journal-title":"Experimental & Molecular Medicine"},{"issue":"1","key":"2025120800592408000_2025.12.01.691650v1.4","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1146\/annurev-virology-010320-061642","article-title":"Within-Host Viral Diversity: A Window into Viral Evolution","volume":"7","year":"2020","journal-title":"Annual Review of Virology"},{"key":"2025120800592408000_2025.12.01.691650v1.5","doi-asserted-by":"publisher","DOI":"10.1016\/j.tim.2018.02.007"},{"issue":"1","key":"2025120800592408000_2025.12.01.691650v1.6","doi-asserted-by":"crossref","first-page":"2834","DOI":"10.1038\/s41467-023-38184-3","article-title":"Enabling accurate and early detection of recently emerged SARS-CoV-2 variants of concern in wastewater","volume":"14","year":"2023","journal-title":"Nature Communications"},{"issue":"7","key":"2025120800592408000_2025.12.01.691650v1.7","doi-asserted-by":"crossref","first-page":"e70133","DOI":"10.1111\/irv.70133","article-title":"Impact of Ventilation on Respiratory Virus Transmission in College Residence Hall Cohorts: Potential for Causal Inference About Mode of Transmission","volume":"19","year":"2025","journal-title":"Influenza and Other Respiratory Viruses"},{"key":"2025120800592408000_2025.12.01.691650v1.8","doi-asserted-by":"publisher","DOI":"10.7717\/peerj.9338"},{"key":"2025120800592408000_2025.12.01.691650v1.9","doi-asserted-by":"publisher","DOI":"10.1101\/gr.277679.123"},{"issue":"4","key":"2025120800592408000_2025.12.01.691650v1.10","first-page":"357","volume-title":"Nature Methods","volume":"9","year":"2012"},{"key":"2025120800592408000_2025.12.01.691650v1.11","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btp324"},{"key":"2025120800592408000_2025.12.01.691650v1.12","doi-asserted-by":"publisher","DOI":"10.1038\/s41576-022-00551-z"},{"key":"2025120800592408000_2025.12.01.691650v1.13","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btw460"},{"issue":"1","key":"2025120800592408000_2025.12.01.691650v1.14","doi-asserted-by":"crossref","first-page":"20444","DOI":"10.1038\/s41598-023-47135-3","article-title":"Evaluating the performance of low-frequency variant calling tools for the detection of variants from short-read deep sequencing data","volume":"13","year":"2023","journal-title":"Scientific Reports"},{"key":"2025120800592408000_2025.12.01.691650v1.15","doi-asserted-by":"publisher","DOI":"10.1038\/ng.1028"},{"issue":"12","key":"2025120800592408000_2025.12.01.691650v1.16","doi-asserted-by":"crossref","first-page":"2529","DOI":"10.1038\/nprot.2016.150","article-title":"Indel variant analysis of short-read sequencing data with Scalpel","volume":"11","year":"2016","journal-title":"Nature Protocols"},{"key":"2025120800592408000_2025.12.01.691650v1.17","doi-asserted-by":"crossref","unstructured":"Pierre Peterlongo , Chlo\u00e9 Riou , Erwan Drezen , and Claire Lemaitre . 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