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Müller, Carolin A., Boemo, Michael A., Spingardi, Paolo, Kessler, Benedikt M., Kriaucionis, Skirmantas, Simpson, Jared T. and Nieduszynski, Conrad A. 
ORCID: https://orcid.org/0000-0003-2001-076X
(2019)
Capturing the dynamics of genome replication on individual ultra-long nanopore sequence reads.
Nature Methods, 16 (5).
pp. 429-436.
ISSN 1548-7091
Abstract
Replication of eukaryotic genomes is highly stochastic, making it difficult to determine the replication dynamics of individual molecules with existing methods. We report a sequencing method for the measurement of replication fork movement on single molecules by detecting nucleotide analog signal currents on extremely long nanopore traces (D-NAscent). Using this method, we detect 5-bromodeoxyuridine (BrdU) incorporated by Saccharomyces cerevisiae to reveal, at a genomic scale and on single molecules, the DNA sequences replicated during a pulse-labeling period. Under conditions of limiting BrdU concentration, D-NAscent detects the differences in BrdU incorporation frequency across individual molecules to reveal the location of active replication origins, fork direction, termination sites, and fork pausing/stalling events. We used sequencing reads of 20–160 kilobases to generate a whole-genome single-molecule map of DNA replication dynamics and discover a class of low-frequency stochastic origins in budding yeast. The D-NAscent software is available at https://github.com/MBoemo/DNAscent.git.
| Item Type: | Article |
|---|---|
| Additional Information: | Funding information: This work was supported by Biotechnology and Biological Sciences Research Council grant BB/N016858/1 (to C.A.M. and C.A.N.) and Wellcome Trust Investigator Award 110064/Z/15/Z (to C.A.N.). J.T.S. is supported by the Ontario Institute for Cancer Research through funds provided by the Government of Ontario and the Government of Canada through Genome Canada and Ontario Genomics (OGI-136). P.S. is funded by a Medical Research Council studentship. P.S. and S.K. are funded by Ludwig Cancer Research. C.A.M is a Queen’s College Extraordinary Junior Research Fellow in Physiology. M.A.B. is a St. Cross College Emanoel Lee Junior Research Fellow. |
| Uncontrolled Keywords: | biotechnology,biochemistry,molecular biology,cell biology ,/dk/atira/pure/subjectarea/asjc/1300/1305 |
| Faculty \ School: | Faculty of Science > School of Biological Sciences |
| Related URLs: | |
| Depositing User: | LivePure Connector |
| Date Deposited: | 07 Sep 2022 14:31 |
| Last Modified: | 14 Jun 2023 14:09 |
| URI: | https://ueaeprints.uea.ac.uk/id/eprint/87854 |
| DOI: | 10.1038/s41592-019-0394-y |
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