Capturing the dynamics of genome replication on individual ultra-long nanopore sequence reads

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

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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
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Depositing User: LivePure Connector
Date Deposited: 07 Sep 2022 14:31
Last Modified: 29 Sep 2022 18:31
URI: https://ueaeprints.uea.ac.uk/id/eprint/87854
DOI: 10.1038/s41592-019-0394-y

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