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Here, we present vAMPirus, a user\u2010friendly, comprehensive, and versatile DNA and RNA virus amplicon sequence analysis program, designed to support investigators in exploring virus amplicon sequencing data and running informed, reproducible analyses. vAMPirus intakes raw virus amplicon libraries and, by default, performs nucleotide\u2010 and amino acid\u2010based analyses to produce results such as sequence abundance information, taxonomic classifications, phylogenies and community diversity metrics. The vAMPirus analytical framework leverages 16 different opensource tools and provides optional approaches that can increase the ratio of biological signal\u2010to\u2010noise and thereby reveal patterns that would have otherwise been masked. Here, we validate the vAMPirus analytical framework and illustrate its implementation as a general virus amplicon sequencing workflow by recapitulating findings from two previously published double\u2010stranded DNA virus datasets. As a case study, we also apply the program to explore the diversity and distribution of a coral reef\u2010associated RNA virus. vAMPirus is streamlined within Nextflow, offering straightforward scalability, standardization and communication of virus lineage\u2010specific analyses. The vAMPirus framework is designed to be adaptable; community\u2010driven analytical standards will continue to be incorporated as the field advances. vAMPirus supports researchers in revealing patterns of virus diversity and population dynamics in nature, while promoting study reproducibility and comparability.<\/jats:p>","DOI":"10.1111\/1755-0998.13978","type":"journal-article","created":{"date-parts":[[2024,5,22]],"date-time":"2024-05-22T10:20:51Z","timestamp":1716373251000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["<scp>vAMPirus<\/scp>: A versatile amplicon processing and analysis program for studying viruses"],"prefix":"10.1111","volume":"24","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3118-5127","authenticated-orcid":false,"given":"Alex J.","family":"Veglia","sequence":"first","affiliation":[{"name":"BioSciences Department Rice University  Houston Texas USA"},{"name":"Department of Biology University of Puerto Rico Mayag\u00fcez  Mayag\u00fcez Puerto Rico USA"},{"name":"EcoAzul  La Parguera Puerto Rico USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6229-3537","authenticated-orcid":false,"given":"Ram\u00f3n E.","family":"Rivera\u2010Vic\u00e9ns","sequence":"additional","affiliation":[{"name":"EcoAzul  La Parguera Puerto Rico USA"},{"name":"Department of Earth and Environmental Sciences, Paleontology &amp; Geobiology Ludwig\u2010Maximilians\u2010Universit\u00e4t M\u00fcnchen  Munich Germany"},{"name":"Department of Sciences and Technology Interamerican University of Puerto Rico at Barranquitas  Barranquitas Puerto Rico USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5083-4570","authenticated-orcid":false,"given":"Carsten G. 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