{"doi":"10.64898/2025.12.22.696044","title":"Improved short nascent strand sequencing (iSNS-seq) enhances DNA replication origin detection and reduces non-origin biases","abstract":"Abstract 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 λ -exonuclease ( λ -exo) digestion of parental DNA. Surprisingly, SNS-seq has never been validated in Saccharomyces cerevisiae where origins have been well characterized genome-wide. We improved the SNS-seq protocol through biochemical optimization and benchmarked its origin-mapping sensitivity and precision with traditional SNS-seq in asynchronous populations of S. cerevisiae . The improved SNS-seq protocol significantly enhanced the enrichment of origin-derived DNA. Strikingly, the 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 λ -exo for origin mapping. Overall, 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.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":586960,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9499,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":573769,"name":"John Urban","orcid":"0000-0001-9270-6569","position":1,"is_corresponding":false},{"id":583341,"name":"Nicola Neretti","orcid":"0000-0002-9552-1663","position":2,"is_corresponding":false},{"id":573773,"name":"Susan A. Gerbi","orcid":"0000-0003-2148-7180","position":3,"is_corresponding":false},{"id":1018273,"name":"Miiko Sokka","orcid":"0000-0002-3091-3754","position":0,"is_corresponding":true}],"reference_count":54,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:59:36.020030Z","pmid":"42244539","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}