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.
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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.
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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.
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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
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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 (2020). https://doi.org/10.1038/s41596-019-0287-7