Double and single stranded detection of 5-methylcytosine and 5-hydroxymethylcytosine with nanopore sequencing

Dominic Oliver Halliwell; Floris Honig; Stefan Bagby; Sandipan Roy; Adele Murrell

doi:10.1038/s42003-025-07681-0

Double and single stranded detection of 5-methylcytosine and 5-hydroxymethylcytosine with nanopore sequencing

Dominic Oliver Halliwell
, Floris Honig
, Stefan Bagby
, Sandipan Roy
, Adele Murrell

Research output: Contribution to journal › Article › peer-review

15 Citations (SciVal)

Abstract

5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are modified versions of cytosine in DNA with roles in regulating gene expression. 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.

Original language	English
Article number	243
Number of pages	1
Journal	Communications Biology
Volume	8
Issue number	1
Early online date	15 Feb 2025
DOIs	https://doi.org/10.1038/s42003-025-07681-0
Publication status	Published - Dec 2025

Bibliographical note

Publisher Copyright:
© The Author(s) 2025.

Data Availability Statement

Raw nanopore machine data in fast5 format has been made available for all murine whole genome sequence experiments on the Sequence Read Archive (SRA) as BioProject PRJNA1144670. Aligned sequence data in BAM file format is also available. Additionally, CpG context modified base detections, as produced by ‘modkit pileup’ are available on the NCBI GEO archive with the accession: GSE279860. These data are limited to CpG positions relative to the mm39 reference genome and are soft-masked. Machine data is not available for the Zymo DNA Methylation Standards; however, these are available as BAM format files under the same BioProject. For the nanopore hMeDIP-seq experiments, data is available in both BAM format under the previously mentioned SRA BioProject, as well as in pod5 format on Zenodo, with record https://doi.org/10.5281/zenodo.14514705. BAM format sequence data used as an input is available as SRR30150148 on the SRA. Narrow peak data, along with direct modified base detections from those peaks, is downloadable under the GEO Series GSE288331. Source data for figures can be downloaded from Figshare (https://doi.org/10.6084/m9.figshare.28287962.v2)92.

Acknowledgements

In addition, the authors would like to thank Sarah Corsi, Nathan Bagby, Marcus Stoiber, Mark Bruce, and Adrien Leger from Oxford Nanopore Technologies for their invaluable support and recommendations. The authors would like to thank Dr. Kim Moorwood of the University of Bath for her support in procuring biological samples for this research.

Funding

This work was supported by funding from the Engineering and Physical Sciences Research Council (EPSRC) [grant number 2598658]. Oxford Nanopore Technologies provided financial support for nanopore sequencing. In addition, the authors would like to thank Sarah Corsi, Nathan Bagby, Marcus Stoiber, Mark Bruce, and Adrien Leger from Oxford Nanopore Technologies for their invaluable support and recommendations. The authors would like to thank Dr. Kim Moorwood of the University of Bath for her support in procuring biological samples for this research. F.H. is supported by the Biotechnology and Biological Sciences Research Council-funded South West Biosciences Doctoral Training Partnership (DTP3: BB/T008741/1) in partnership with CASE partner bit.bio.

Funders	Funder number
Engineering and Physical Sciences Research Council	2598658
Biotechnology and Biological Sciences Research Council-funded South West Biosciences	BB/T008741/1

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Cite this

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Double and single stranded detection of 5-methylcytosine and 5-hydroxymethylcytosine with nanopore sequencing

Abstract

Bibliographical note

Data Availability Statement

Acknowledgements

Funding

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