{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,10,21]],"date-time":"2026-10-21T20:05:09Z","timestamp":1792613109635,"version":"4.5.0"},"posted":{"date-parts":[[2026,9,19]]},"group-title":"Microbiology","reference-count":26,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,9,19]],"date-time":"2026-09-19T00:00:00Z","timestamp":1789776000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,9,30]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Reference genomes are often treated as faithful representations of experimentally validated viral genomes, yet the relationship between historically curated reference sequences and infectivity is rarely tested experimentally. Here, we developed a cDNA-based reconstruction platform for the canonical RNA phage MS2 and used it to compare the current NCBI reference genome (RefSeq) with closely related published isolate sequences. We found that isolate-derived sequences reproducibly yielded infectious phage, whereas the current MS2 RefSeq-derived construct did not, showing that the present reference does not represent a single experimentally validated infectious genome but instead reflects sequence curation across multiple studies. We then compared conventional and AI-enabled approaches to identify minimal changes that restore infectivity to MS2 RefSeq; a human experimentalist correctly prioritized corrective changes, whereas the genome language model Evo2 did not. We also observed that closely related corrected reference-derived constructs showed a ~4-log difference in phage output, and subsequent analysis indicated that this difference was associated with an apparent replicase frameshift in the lower-output background. This suggests that the low output construct class represents rare mutations from genomes that are one mutational step away from true function, rather than uniform function of the dominant construct population. A complementary cell-free assay provided a lower-background orthogonal readout of construct-level function, yielding ~1 x10\n                  <jats:sup>6<\/jats:sup>\n                  PFU\/mL from the high-output background within 2 hours while showing no detectable recovery from the low-output background. Together, these results establish a robust platform for RNA phage reconstruction and raise the possibility that historical reference genomes, especially for RNA viruses, may not always remain faithful to experimentally validated biological function. More broadly, these findings underscore the need to verify the infectivity of reference genomes, particularly when they were assembled non-contiguously or shaped by cumulative human curation. They also highlight the importance of clearly distinguishing historically curated reference sequences from experimentally validated infectious genomes when such data are used to train or evaluate AI\/ML models.\n                <\/jats:p>","DOI":"10.64898\/2026.09.18.752701","type":"posted-content","created":{"date-parts":[[2026,9,20]],"date-time":"2026-09-20T03:10:12Z","timestamp":1789873812000},"source":"Crossref","is-referenced-by-count":2,"title":["A reference genome without a virus: cDNA reconstruction reveals the provenance and function of the MS2 phage sequence"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6960-4393","authenticated-orcid":false,"given":"Eric","family":"Small","sequence":"first","affiliation":[{"name":"Environmental Systems Biology, Sandia National Laboratories, Albuquerque NM, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Greyson","family":"Lasley","sequence":"additional","affiliation":[{"name":"Environmental Systems Biology, Sandia National Laboratories, Albuquerque NM, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emily","family":"Layton","sequence":"additional","affiliation":[{"name":"Environmental Systems Biology, Sandia National Laboratories, Albuquerque NM, USA"},{"name":"Department of Biology, Indiana University, Bloomington IN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alissa","family":"Wiwi","sequence":"additional","affiliation":[{"name":"Environmental Systems Biology, Sandia National Laboratories, Albuquerque NM, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-4862-4283","authenticated-orcid":false,"given":"Devyn","family":"Del Curto","sequence":"additional","affiliation":[{"name":"Environmental Systems Biology, Sandia National Laboratories, Albuquerque NM, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Laura D.","family":"Weinstock","sequence":"additional","affiliation":[{"name":"Systems Research & Analysis, Sandia National Laboratories, Livermore, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jirapat","family":"Thongchol","sequence":"additional","affiliation":[{"name":"Center for Phage Technology, Department of Biochemistry and Biophysics, Texas A&M University, College Station TX, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Junjie","family":"Zhang","sequence":"additional","affiliation":[{"name":"Center for Phage Technology, Department of Biochemistry and Biophysics, Texas A&M University, College Station TX, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8387-9273","authenticated-orcid":false,"given":"Jesse","family":"Cahill","sequence":"additional","affiliation":[{"name":"Environmental Systems Biology, Sandia National Laboratories, Albuquerque NM, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"issue":"10","key":"2026100518400782000_2026.09.18.752701v2.1","first-page":"1985","article-title":"Recent advances in structural studies of single-stranded RNA bacteriophages","volume":"2023","year":"2023","journal-title":"Viruses"},{"issue":"7","key":"2026100518400782000_2026.09.18.752701v2.2","first-page":"1225","article-title":"The asymmetric structure of an icosahedral virus bound to its receptor suggests a mechanism for genome release","volume":"2013","year":"2013","journal-title":"Structure"},{"issue":"2","key":"2026100518400782000_2026.09.18.752701v2.3","first-page":"431","article-title":"Direct Evidence for Packaging Signal-Mediated Assembly of Bacteriophage MS2","volume":"2016","year":"2016","journal-title":"Journal of Molecular Biology"},{"issue":"1","key":"2026100518400782000_2026.09.18.752701v2.4","first-page":"3130","article-title":"Structural basis for the adsorption of a 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interaction with an RNA virus","volume":"2024","year":"2024","journal-title":"Science Advances"},{"issue":"3","key":"2026100518400782000_2026.09.18.752701v2.18","first-page":"509","article-title":"Interrupting the template strand of the T7 promoter facilitates translocation of the DNA during initiation, reducing transcript slippage and the release of abortive products","volume":"2001","year":"2001","journal-title":"Journal of Molecular Biology"},{"issue":"1","key":"2026100518400782000_2026.09.18.752701v2.19","first-page":"99","article-title":"Nanopore direct RNA sequencing (DRS) of MS2 bacteriophages in E. coli throughout its life cycles reveals a complex transcriptional activity to control and maintain its growth","volume":"2026","year":"2026","journal-title":"Virology Journal"},{"key":"2026100518400782000_2026.09.18.752701v2.20","first-page":"1718418","article-title":"Assessment of the phi6 lysis system using genetic complementation and heterologous expression in Escherichia coli and 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