Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

DNA copy-number measurement of genome replication dynamics by high-throughput sequencing: the sort-seq, sync-seq and MFA-seq family

Abstract

Genome replication follows a defined temporal programme that can change during cellular differentiation and disease onset. DNA replication results in an increase in DNA copy number that can be measured by high-throughput sequencing. Here we present a protocol to determine genome replication dynamics using DNA copy-number measurements. Cell populations can be obtained in three variants of the method. First, sort-seq reveals the average replication dynamics across S phase in an unperturbed cell population; FACS is used to isolate replicating and non-replicating subpopulations from asynchronous cells. Second, sync-seq measures absolute replication time at specific points during S phase using a synchronized cell population. Third, marker frequency analysis can be used to reveal the average replication dynamics using copy-number analysis in any proliferating asynchronous cell culture. These approaches have been used to reveal genome replication dynamics in prokaryotes, archaea and a wide range of eukaryotes, including yeasts and mammalian cells. We have found this approach straightforward to apply to other organisms and highlight example studies from across the three domains of life. Here we present a Saccharomyces cerevisiae version of the protocol that can be performed in 7–10 d. It requires basic molecular and cellular biology skills, as well as a basic understanding of Unix and R.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Workflow overview.
Fig. 2: Cell sorting gates.
Fig. 3: Sync-seq timecourse.
Fig. 4: Library preparation.
Fig. 5: Anticipated results.
Fig. 6: Chromosomal aberrations.

Data availability

All data generated or analyzed during this study are publicly available from the NCBI GEO functional genomics data repository with the following accession numbers: GSE42243, GSE48212 (Fig. 5); GSE135178 (Fig. 6).

Code availability

The custom bash script required for the analysis, as well as the script to download and analyze example data, are available from GitHub (https://github.com/DNAReplicationLab/localMapper/). The R package Repliscope described here is available from CRAN (https://cran.r-project.org/web/packages/Repliscope/) and GitHub (https://github.com/DNAReplicationLab/Repliscope/). We have also provided an official Ubuntu Desktop 18.04 LTS installation disk image with all the software required for the analysis (https://ln1.path.ox.ac.uk/groups/nieduszynski/Replibuntu/Replibuntu-18.04.0-amd64.iso.gz). The code in this manuscript has been peer reviewed.

References

  1. 1.

    Fragkos, M., Ganier, O., Coulombe, P. & Mechali, M. DNA replication origin activation in space and time. Nat. Rev. Mol. Cell. Biol. 16, 360–374 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Newman, T. J., Mamun, M. A., Nieduszynski, C. A. & Blow, J. J. Replisome stall events have shaped the distribution of replication origins in the genomes of yeasts. Nucleic Acids Res. 41, 9705–9718 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Rhind, N. & Gilbert, D. M. DNA replication timing. Cold Spring Harb. Perspect. Biol. 5, a010132 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Müller, C. A. et al. The dynamics of genome replication using deep sequencing. Nucleic Acids Res. 42, e3 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Yabuki, N., Terashima, H. & Kitada, K. Mapping of early firing origins on a replication profile of budding yeast. Genes Cells 7, 781–789 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Raghuraman, M. K. et al. Replication dynamics of the yeast genome. Science 294, 115–121 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Peace, J. M., Villwock, S. K., Zeytounian, J. L., Gan, Y. & Aparicio, O. M. Quantitative BrdU immunoprecipitation method demonstrates that Fkh1 and Fkh2 are rate-limiting activators of replication origins that reprogram replication timing in G1 phase. Genome Res. 26, 365–375 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Sekedat, M. D. et al. GINS motion reveals replication fork progression is remarkably uniform throughout the yeast genome. Mol. Syst. Biol. 6, 353 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Hawkins, M. et al. High-resolution replication profiles define the stochastic nature of genome replication initiation and termination. Cell Rep. 5, 1132–1141 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Claycomb, J. M. & Orr-Weaver, T. L. Developmental gene amplification: insights into DNA replication and gene expression. Trends Genet. 21, 149–162 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Koren, A., Soifer, I. & Barkai, N. MRC1-dependent scaling of the budding yeast DNA replication timing program. Genome Res. 20, 781–790 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Müller, C. A. & Nieduszynski, C. A. Conservation of replication timing reveals global and local regulation of replication origin activity. Genome Res. 22, 1953–1962 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Skovgaard, O., Bak, M., Lobner-Olesen, A. & Tommerup, N. Genome-wide detection of chromosomal rearrangements, indels, and mutations in circular chromosomes by short read sequencing. Genome Res. 21, 1388–1393 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Hawkins, M., Malla, S., Blythe, M. J., Nieduszynski, C. A. & Allers, T. Accelerated growth in the absence of DNA replication origins. Nature 503, 544–547 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Samson, R. Y. et al. Specificity and function of archaeal DNA replication initiator proteins. Cell Rep. 3, 485–496 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Agier, N. et al. The evolution of the temporal program of genome replication. Nat. Commun. 9, 2199 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Koren, A. et al. Genetic variation in human DNA replication timing. Cell 159, 1015–1026 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Batrakou, D. G., Heron, E. D. & Nieduszynski, C. A. Rapid high-resolution measurement of DNA replication timing by droplet digital PCR. Nucleic Acids Res. 46, e112 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Shen, Y. et al. Deep functional analysis of synII, a 770-kilobase synthetic yeast chromosome. Science 355, eaaf4791 https://doi.org/10.1126/science.aaf4791 (2017).

  20. 20.

    Annaluru, N. et al. Total synthesis of a functional designer eukaryotic chromosome. Science 344, 55–58 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Müller, C. A. & Nieduszynski, C. A. DNA replication timing influences gene expression level. J. Cell Biol. 216, 1907–1914 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Natsume, T. et al. Kinetochores coordinate pericentromeric cohesion and early DNA replication by Cdc7-Dbf4 kinase recruitment. Mol. Cell 50, 661–674 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Kedziora, S. et al. Rif1 acts through Protein Phosphatase 1 but independent of replication timing to suppress telomere extension in budding yeast. Nucleic Acids Res. 46, 3993–4003 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Foss, E. J. et al. SIR2 suppresses replication gaps and genome instability by balancing replication between repetitive and unique sequences. Proc. Natl Acad. Sci. USA 114, 552–557 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Voichek, Y., Bar-Ziv, R. & Barkai, N. Expression homeostasis during DNA replication. Science 351, 1087–1090 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Rudolph, C. J., Upton, A. L., Stockum, A., Nieduszynski, C. A. & Lloyd, R. G. Avoiding chromosome pathology when replication forks collide. Nature 500, 608–611 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Tanaka, S., Nakato, R., Katou, Y., Shirahige, K. & Araki, H. Origin association of Sld3, Sld7, and Cdc45 proteins is a key step for determination of origin-firing timing. Curr. Biol. 21, 2055–2063 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Green, B. M., Finn, K. J. & Li, J. J. Loss of DNA replication control is a potent inducer of gene amplification. Science 329, 943–946 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Tanny, R. E., MacAlpine, D. M., Blitzblau, H. G. & Bell, S. P. Genome-wide analysis of re-replication reveals inhibitory controls that target multiple stages of replication initiation. Mol. Biol. Cell 17, 2415–2423 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Kramara, J., Osia, B. & Malkova, A. Break-induced replication: the where, the why, and the how. Trends Genet. 34, 518–531 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Alvino, G. M. et al. Replication in hydroxyurea: it’s a matter of time. Mol. Cell. Biol. 27, 6396–6406 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Macheret, M. & Halazonetis, T. D. Monitoring early S-phase origin firing and replication fork movement by sequencing nascent DNA from synchronized cells. Nat. Protoc. 14, 51–67 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Macheret, M. & Halazonetis, T. D. Intragenic origins due to short G1 phases underlie oncogene-induced DNA replication stress. Nature 555, 112–116 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Marchal, C. et al. Genome-wide analysis of replication timing by next-generation sequencing with E/L Repli-seq. Nat. Protoc. 13, 819–839 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Siow, C. C., Nieduszynska, S. R., Müller, C. A. & Nieduszynski, C. A. OriDB, the DNA replication origin database updated and extended. Nucleic Acids Res. 40, D682–D686 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Prioleau, M. N. & MacAlpine, D. M. DNA replication origins—where do we begin? Genes Dev. 30, 1683–1697 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Jinks-Robertson, S. & Klein, H. L. Ribonucleotides in DNA: hidden in plain sight. Nat. Struct. Mol. Biol. 22, 176–178 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Petryk, N. et al. Replication landscape of the human genome. Nat. Commun. 7, 10208 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    McGuffee, S. R., Smith, D. J. & Whitehouse, I. Quantitative, genome-wide analysis of eukaryotic replication initiation and termination. Mol. Cell 50, 123–135 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Feng, W. et al. Genomic mapping of single-stranded DNA in hydroxyurea-challenged yeasts identifies origins of replication. Nat. Cell Biol. 8, 148–155 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Tiengwe, C. et al. Genome-wide analysis reveals extensive functional interaction between DNA replication initiation and transcription in the genome of Trypanosoma brucei. Cell Rep. 2, 185–197 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Donaldson, A. D. & Nieduszynski, C. A. Genome-wide analysis of DNA replication timing in single cells: Yes! We’re all individuals. Genome Biol. 20, 111 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  43. 43.

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Haase, S. B. & Reed, S. I. Improved flow cytometric analysis of the budding yeast cell cycle. Cell Cycle 1, 132–136 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Daigaku, Y. et al. A global profile of replicative polymerase usage. Nat. Struct. Mol. Biol. 22, 192–198 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Xu, J. et al. Genome-wide identification and characterization of replication origins by deep sequencing. Genome Biol. 13, R27 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Heichinger, C., Penkett, C. J., Bahler, J. & Nurse, P. Genome-wide characterization of fission yeast DNA replication origins. EMBO J. 25, 5171–5179 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Agier, N., Romano, O. M., Touzain, F., Cosentino Lagomarsino, M. & Fischer, G. The spatiotemporal program of replication in the genome of Lachancea kluyveri. Genome Biol. Evol. 5, 370–388 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Di Rienzi, S. C. et al. Maintaining replication origins in the face of genomic change. Genome Res. 22, 1940–1952 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Kim, H. S. Genome-wide function of MCM-BP in Trypanosoma brucei DNA replication and transcription. Nucleic Acids Res. 47, 634–647 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. 55.

    Devlin, R. et al. Mapping replication dynamics in Trypanosoma brucei reveals a link with telomere transcription and antigenic variation. Elife 5, e12765 https://doi.org/10.7554/eLife.12765 (2016).

  56. 56.

    Marques, C. A., Dickens, N. J., Paape, D., Campbell, S. J. & McCulloch, R. Genome-wide mapping reveals single-origin chromosome replication in Leishmania, a eukaryotic microbe. Genome Biol. 16, 230 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. 57.

    Lundgren, M., Andersson, A., Chen, L., Nilsson, P. & Bernander, R. Three replication origins in Sulfolobus species: synchronous initiation of chromosome replication and asynchronous termination. Proc. Natl Acad. Sci. USA 101, 7046–7051 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Woodfine, K. et al. Replication timing of the human genome. Hum. Mol. Genet. 13, 191–202 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. 59.

    de Moura, A. P., Retkute, R., Hawkins, M. & Nieduszynski, C. A. Mathematical modelling of whole chromosome replication. Nucleic Acids Res. 38, 5623–5633 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank Amanda Williams and Becky Busby (Zoology Sequencing Facility) for help with the NextSeq 500, and Michal Maj and Line Eriksen (Sir William Dunn School Flow Cytometry Facility) for their help with FACS.

Author information

Affiliations

Authors

Contributions

All authors wrote and edited the manuscript.

Corresponding author

Correspondence to Conrad A. Nieduszynski.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Hawkins, M. et al. Nature 503, 544–547 (2013): https://doi.org/10.1038/nature12650

Müller, C. A. et al. Nucleic Acids Res. 42, e3 (2014): https://doi.org/10.1093/nar/gkt878

Müller, C. A. & Nieduszynski, C. A. J. Cell Biol. 216, 1907–1914 (2017): https://doi.org/10.1083/jcb.201701061

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Batrakou, D.G., Müller, C.A., Wilson, R.H.C. et al. DNA copy-number measurement of genome replication dynamics by high-throughput sequencing: the sort-seq, sync-seq and MFA-seq family. Nat Protoc 15, 1255–1284 (2020). https://doi.org/10.1038/s41596-019-0287-7

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing