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Using whole genomic DNA from mouse cerebellum, we benchmark 5mC and 5hmC detection by Oxford Nanopore Technologies sequencing against other standard techniques. In addition, we assess the ability of duplex base-calling to study strand asymmetric modification. Nanopore detection of 5mC and 5hmC is accurate relative to compared techniques and opens means of studying these modifications. Strand asymmetric modification is widespread across the genome but reduced at imprinting control regions and CTCF binding sites in mouse cerebellum. Here we demonstrate the unique ability of nanopore sequencing to improve the resolution and detail of cytosine modification mapping.<\/jats:p>","DOI":"10.1038\/s42003-025-07681-0","type":"journal-article","created":{"date-parts":[[2025,2,15]],"date-time":"2025-02-15T12:10:26Z","timestamp":1739621426000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":34,"title":["Double and single stranded detection of 5-methylcytosine and 5-hydroxymethylcytosine with nanopore sequencing"],"prefix":"10.1038","volume":"8","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2848-7888","authenticated-orcid":false,"given":"Dominic Oliver","family":"Halliwell","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6006-7376","authenticated-orcid":false,"given":"Floris","family":"Honig","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2302-9511","authenticated-orcid":false,"given":"Stefan","family":"Bagby","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2898-0659","authenticated-orcid":false,"given":"Sandipan","family":"Roy","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2647-3511","authenticated-orcid":false,"given":"Adele","family":"Murrell","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,2,15]]},"reference":[{"key":"7681_CR1","doi-asserted-by":"publisher","first-page":"172","DOI":"10.1016\/j.pharmthera.2018.11.001","volume":"195","author":"A Unnikrishnan","year":"2019","unstructured":"Unnikrishnan, A. et al. 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