{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,5]],"date-time":"2026-09-05T03:13:19Z","timestamp":1788577999733,"version":"build-2803163510"},"posted":{"date-parts":[[2025,12,24]]},"group-title":"Genomics","reference-count":59,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,12,24]],"date-time":"2025-12-24T00:00:00Z","timestamp":1766534400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["GM121455"],"award-info":[{"award-number":["GM121455"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,9,2]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Identifying DNA replication origins in human and other metazoan genomes has been challenging, as highlighted by the fact that various methods for mapping them have produced conflicting results. A popular method, short nascent strand sequencing (SNS-seq), enriches newly replicated short single-stranded DNA by size selection and\n                  <jats:italic>\u03bb<\/jats:italic>\n                  -exonuclease (\n                  <jats:italic>\u03bb<\/jats:italic>\n                  -exo) digestion of parental DNA. Surprisingly, SNS-seq has never been validated in\n                  <jats:italic>Saccharomyces cerevisiae<\/jats:italic>\n                  where origins have been well characterized genome-wide. Here we improved the SNS-seq protocol through biochemical optimization and benchmarked its origin-mapping sensitivity and precision against traditional SNS-seq in asynchronous populations of\n                  <jats:italic>S. cerevisiae<\/jats:italic>\n                  , a genetically tractable system that allows direct comparison against a well-defined set of confirmed origins. Relative to traditional SNS-seq, the improved protocol substantially increased enrichment of origin-derived DNA. Strikingly, traditional SNS-seq failed to detect known origins and instead enriched non-origin DNA, likely arising from RNA:DNA hybrids. These findings have important implications for the interpretation of previously published datasets that rely on\n                  <jats:italic>\u03bb<\/jats:italic>\n                  -exo for origin mapping, and provide a proof-of-concept benchmark for extending this improved protocol to metazoan systems. Furthermore, our biochemical and genomic analyses help unravel the mystery of the inconsistencies between SNS-seq and other techniques used to map DNA replication origins genome-wide.\n                <\/jats:p>","DOI":"10.64898\/2025.12.22.696044","type":"posted-content","created":{"date-parts":[[2025,12,24]],"date-time":"2025-12-24T18:30:19Z","timestamp":1766601019000},"source":"Crossref","is-referenced-by-count":0,"title":["Improved short nascent strand sequencing (iSNS-seq) enhances DNA replication origin detection and reduces non-origin biases"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3091-3754","authenticated-orcid":false,"given":"Miiko","family":"Sokka","sequence":"first","affiliation":[{"name":"Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, 185 Meeting Street, Providence, Rhode Island, 02912, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9270-6569","authenticated-orcid":false,"given":"John M.","family":"Urban","sequence":"additional","affiliation":[{"name":"Department of Embryology, Carnegie Institution for Science, 3520 San Martin Drive, Baltimore, Maryland, 21218, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9552-1663","authenticated-orcid":false,"given":"Nicola","family":"Neretti","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, 185 Meeting Street, Providence, Rhode Island, 02912, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2148-7180","authenticated-orcid":false,"given":"Susan A.","family":"Gerbi","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, 185 Meeting Street, Providence, Rhode Island, 02912, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026090420000498000_2025.12.22.696044v3.1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-biochem-072321-110228"},{"key":"2026090420000498000_2025.12.22.696044v3.2","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2022.12.024"},{"key":"2026090420000498000_2025.12.22.696044v3.3","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.bi.62.070193.000333"},{"key":"2026090420000498000_2025.12.22.696044v3.4","doi-asserted-by":"crossref","unstructured":"Petryk, N. , Kahli, M. , D\u2019Aubenton-Carafa, Y. , Jaszczyszyn, Y. , Shen, Y. , Silvain, M. , Thermes, C. , Chen, C. L. , and Hyrien, O. (2016) Replication Landscape of the Human Genome. Nat. Comm., 7.","DOI":"10.1038\/ncomms10208"},{"key":"2026090420000498000_2025.12.22.696044v3.5","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2021.05.024"},{"key":"2026090420000498000_2025.12.22.696044v3.6","doi-asserted-by":"crossref","first-page":"RP88087","DOI":"10.7554\/eLife.88087","article-title":"Identification of 1600 Replication Origins in S. Cerevisiae","volume":"12","year":"2024","journal-title":"eLife"},{"key":"2026090420000498000_2025.12.22.696044v3.7","doi-asserted-by":"crossref","unstructured":"Urban, J. M. , Foulk, M. S. , Casella, C. , and Gerbi, S. A. (2015) The Hunt for Origins of DNA Replication in Multicellular Eukaryotes. 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