Abstract
Current approaches to mapping fork progression in the human genome suffer from drastically low throughput. Here, we introduce ForkML, a nanopore sequencing-based method automatically positioning thousands of individual fork velocities by tracking BrdU incorporation into replicating DNA after double pulse-labelling of asynchronous cells. ForkML recovers known human fork speed, accurately detects replication stress, and, crucially, connects replication dynamics to genomic and chromatin contexts, exposing fork slowdown in early-replicating transcribed regions.
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Data availability
Nanopore sequencing data generated in this study have been deposited in the ENA database under accession code PRJEB90580. HCT116 GLOE-seq data from ref. 10 used in this study are available in NCBI’s BioProject database under accession code PRJNA554350 (GSM4305465 and GSM4305466). HCT116 high-resolution Repli-seq data from ref. 34 used in this study are available in NCBI’s Gene Expression Omnibus repository under accession code GSE137764 (supplementary file GSE137764_HCT_GaussiansGSE137764_mooth_scaled_autosome.mat.gz). HCT116 chromatin subcompartment data from ref. 35 used in this study are available on GitHub (https://github.com/mirnylab/heterochromatin-paper) (HCT116_Unsynchronized.hg38.50000.clusters.E1-E9.chr1-22.kmeans8_5.labeled.dense.bed file). Additional datasets, including training files (for R9 and R10 chemistries), BrdU basecalling model (for R9 chemistry) and fork detection (for R9 and R10 chemistries) models, as well as processed external datasets, have been deposited on Zenodo (https://doi.org/10.5281/zenodo.15706384) Source data are available at https://github.com/touala/ForkML38.
Code availability
ForkML software is available on GitHub (https://github.com/touala/ForkML)38. Custom R scripts for leading/lagging strand synthesis rate statistical analysis can be accessed at https://gitlab.pasteur.fr/gmillot/anova_contrasts/-/tree/v12.8.
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Acknowledgements
The authors thank Magali Hennion for fruitful discussions, Laurent Lacroix for helpful discussions and critical reading of the manuscript, IBENS GenomiqueENS facility for their assistance with nanopore sequencing and IBENS IT platform and BioClust computing cluster (Labex Memolife) for data management. This work was supported by grants from Fondation pour la Recherche Médicale [FRM EQU202203014910 to O.H.], Agence Nationale pour la Recherche [NanoPoRep ANR-18-CE45-0002, HUDROR ANR-19-CE12-0028 and SMAHGR ANR-23-CE12-0021 to B.A. and O.H.] and Région Île-de-France [DIM1Health 2019 grant to the project EpiK to P.B.A. for the LC/MS-MS equipment]. V.R. was supported by fellowships from the Ministère de l’Enseignement Supérieur et de la Recherche and Fondation pour la Recherche médicale [FRM FDT202404018224].
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V.R., D.C. and F.P. performed the experiments and nanopore sequencing. A.T., D.C. and E.J. performed the computational studies. J.M.A. implemented BrdU detection with ONT’s Megalodon programme. G.A.M. and A.T. performed the statistical analyses. F.B. and P.B.A. carried out mass spectrometry analysis. A.T., B.L.T., D.C., V.R., B.A. and O.H. analysed the data. B.L.T. designed the project, supervised the study and wrote the manuscript with inputs from the other authors.
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Rojat, V., Ciardo, D., Tourancheau, A. et al. Automated mapping of DNA replication fork progression in human cells with ForkML. Nat Commun (2026). https://doi.org/10.1038/s41467-026-68750-4
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DOI: https://doi.org/10.1038/s41467-026-68750-4
