Automated mapping of DNA replication fork progression in human cells with ForkML

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.

1. Saxena, S. & Zou, L. Hallmarks of DNA replication stress. Mol. Cell 82, 2298–2314 (2022).

   Google Scholar 

2. Wang, N., Xu, S. & Egli, D. Replication stress in mammalian embryo development, differentiation, and reprogramming. Trends Cell Biol. 33, 872–886 (2023).

   Google Scholar 

3. Hwang, Y. et al. Global increase in replication fork speed during a p57(KIP2)-regulated erythroid cell fate switch. Sci. Adv. 3, e1700298 (2017).

   Google Scholar 

4. Nakatani, T. et al. DNA replication fork speed underlies cell fate changes and promotes reprogramming. Nat. Genet. 54, 318–327 (2022).

   Google Scholar 

5. Do, B. T. et al. Nucleotide depletion promotes cell fate transitions by inducing DNA replication stress. Dev. Cell 59, 2203–2221 e2215 (2024).

   Google Scholar 

6. Wang, J. et al. DNA replication fork speed acts as a pacer in cortical neurogenesis. Nat. Commun. 16, 10121 (2025).

   Google Scholar 

7. Techer, H. et al. Replication dynamics: biases and robustness of DNA fiber analysis. J. Mol. Biol. 425, 4845–4855 (2013).

   Google Scholar 

8. Hennion, M. et al. FORK-seq: replication landscape of the Saccharomyces cerevisiae genome by nanopore sequencing. Genome Biol. 21, 125 (2020).

   Google Scholar 

9. Theulot, B. et al. Genome-wide mapping of individual replication fork velocities using nanopore sequencing. Nat. Commun. 13, 3295 (2022).

   Google Scholar 

10. Sriramachandran, A. M. et al. Genome-wide nucleotide-resolution mapping of DNA replication patterns, single-strand breaks, and lesions by GLOE-seq. Mol. Cell 78, 975–985 e977 (2020).

    Google Scholar 

11. Petermann, E., Helleday, T. & Caldecott, K. W. Claspin promotes normal replication fork rates in human cells. Mol. Biol. Cell 19, 2373–2378 (2008).

    Google Scholar 

12. Fu, H. et al. The DNA repair endonuclease Mus81 facilitates fast DNA replication in the absence of exogenous damage. Nat. Commun. 6, 6746 (2015).

    Google Scholar 

13. Jones, M. J. K. et al. A high-resolution, nanopore-based artificial intelligence assay for DNA replication stress in human cancer cells. Nat. Commun. 16, 7732 (2025).

    Google Scholar 

14. van den Berg, J. et al. Quantifying DNA replication speeds in single cells by scEdU-seq. Nat. Methods 21, 1175–1184 (2024).

    Google Scholar 

15. Wang, W. et al. Genome-wide mapping of human DNA replication by optical replication mapping supports a stochastic model of eukaryotic replication. Mol. Cell 81, 2975–2988 e2976 (2021).

    Google Scholar 

16. Blin, M. et al. DNA molecular combing-based replication fork directionality profiling. Nucleic Acids Res. 49, e69 (2021).

    Google Scholar 

17. Carrington, J. T. et al. Most human DNA replication initiation is dispersed throughout the genome with only a minority within previously identified initiation zones. Genome Biol. 26, 122 (2025).

    Google Scholar 

18. Muller, C. A. et al. Capturing the dynamics of genome replication on individual ultra-long nanopore sequence reads. Nat. Methods 16, 429–436 (2019).

    Google Scholar 

19. Claussin, C., Vazquez, J. & Whitehouse, I. Single-molecule mapping of replisome progression. Mol. Cell 82, 1372–1382 e1374 (2022).

    Google Scholar 

20. Theulot, B. et al. Telomere-to-telomere DNA replication timing profiling using single-molecule sequencing with Nanotiming. Nat. Commun. 16, 242 (2025).

    Google Scholar 

21. Thiyagarajan, S., Rogers, A. M., Muller, C. A. & Nieduszynski, C. A. Single-molecule landscape of DNA replication pausing. Preprint at https://doi.org/10.1101/2025.08.14.670160 (2025).

22. Cheng, T. et al. Transcription-replication conflict resolution by nuclear RNA interference. Mol. Cell 85, 3930–3946 e3935 (2025).

    Google Scholar 

23. Díez-Santos, I. et al. Single-molecule sequencing maps replication dynamics across the fission yeast genome, including centromeres. Preprint at https://doi.org/10.1101/2025.07.16.665067 (2025).

24. Totanes, F. I. G. et al. A genome-wide map of DNA replication at single-molecule resolution in the malaria parasite Plasmodium falciparum. Nucleic Acids Res. 51, 2709–2724 (2023).

    Google Scholar 

25. Castellano, C. M. et al. The genetic landscape of origins of replication in P. falciparum. Nucleic Acids Res. 52, 660–676 (2024).

    Google Scholar 

26. Totanes, F. I. G. et al. DNA replication dynamics are associated with genome composition in Plasmodium species. Nucleic Acids Res. 53, gkaf111 (2025).

27. de Oliveira Vitarelli, M. et al. Integrating high-throughput analysis to create an atlas of replication origins in Trypanosoma cruzi in the context of genome structure and variability. mBio 15, e0031924 (2024).

    Google Scholar 

28. Damasceno, J. D. et al. Leishmania major chromosomes are replicated from a single high-efficiency locus supplemented by thousands of lower efficiency initiation events. Cell Rep. 44, 116094 (2025).

    Google Scholar 

29. Han, D., Shepherd, C., Benton, M. L. & Nordman, J. T. Nanopore-based sequencing of active DNA replication reveals key principles of metazoan replication fork progression, origin and termination sites. Preprint at https://doi.org/10.1101/2025.09.26.678856 (2025).

30. Jaworski, J. J. et al. ecDNA replication is disorganized and vulnerable to replication stress. Nucleic Acids Res. 53, gkaf711 (2025).

31. Nurk, S. et al. The complete sequence of a human genome. Science 376, 44–53 (2022).

    Google Scholar 

32. R Core Team. R: a language and environment for statistical computing. R Foundation for Statistical Computing. (2025).

33. Ronneberger, O., Fischer, P. & Brox, T. U-Net: convolutional networks for biomedical image segmentation. Preprint at https://doi.org/10.48550/arXiv.1505.04597 (2015).

34. Zhao, P. A., Sasaki, T. & Gilbert, D. M. High-resolution Repli-Seq defines the temporal choreography of initiation, elongation and termination of replication in mammalian cells. Genome Biol. 21, 76 (2020).

    Google Scholar 

35. Spracklin, G. et al. Diverse silent chromatin states modulate genome compartmentalization and loop extrusion barriers. Nat. Struct. Mol. Biol. 30, 38–51 (2023).

    Google Scholar 

36. Hinrichs, A. S. et al. The UCSC Genome Browser Database: update 2006. Nucleic Acids Res. 34, D590–D598 (2006).

    Google Scholar 

37. Kirstein, N. et al. Human ORC/MCM density is low in active genes and correlates with replication time but does not delimit initiation zones. eLife 10, e62161 (2021).

38. Tourancheau, A. touala/ForkML: updated GitHub on 20251223 (v0.2.0). Zenodo, https://doi.org/10.5281/zenodo.18038657 (2025).

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Author notes

1. Jean-Michel Arbona

   Present address: IBDM, UMR7288, Case 907, Parc Scientifique de Luminy, Marseille, France

2. These authors contributed equally: Victoria Rojat, Diletta Ciardo, Alan Tourancheau.

Authors and Affiliations

1. IBENS, Département de biologie, École Normale Supérieure, Université PSL, CNRS, INSERM, Paris, France

   Victoria Rojat, Diletta Ciardo, Alan Tourancheau, Florence Proux, Etienne Jean, Olivier Hyrien & Benoît Le Tallec

2. Laboratoire de Biologie et Modélisation de la Cellule, École Normale Supérieure de Lyon, CNRS, UMR5239, INSERM, U1293, Université Claude Bernard Lyon 1, Lyon, France

   Jean-Michel Arbona

3. CNRS, ENS de Lyon, LPENSL, UMR5672, Lyon, France

   Benjamin Audit

4. Bioinformatics and Biostatistics Hub, Institut Pasteur, Université Paris Cité, Paris, France

   Gael A. Millot

5. Epigenetic Chemical Biology EpiCBio, Institut Pasteur, CNRS UMR3523 Chem4Life, Université Paris Cité, Paris, France

   Frédéric Bonhomme & Paola B. Arimondo

Contributions

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.

Corresponding author

Correspondence to Benoît Le Tallec.

Competing interests

The authors declare no competing interests.

Peer review information

Nature Communications thanks Jeroen van den Berg and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

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