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

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.

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Fig. 1: Nanopore sequencing can distinguish thymidine from analogs.
Fig. 2: BrdU can be distinguished from thymidine in genomic DNA.
Fig. 3: Single-molecule detection of BrdU on nascent DNA.
Fig. 4: Single-molecule detection of replication dynamics.

Data availability

Raw and processed Illumina and MinION data are available from NCBI GEO under accession number GSE121941.

Code availability

The D-NAscent software can be accessed at https://github.com/MBoemo/DNAscent.git.

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Acknowledgements

We are grateful to J. Diffley (Francis Crick Institute, UK) and E. Schwob (Institut de Génétique Moléculaire de Montpellier, France) for kindly providing strains; A. Williams and R. Busby for Illumina NextSeq support; M. Maj for assistance with flow cytometry; J. Caesar for IT infrastructure support; and Microsoft and NVIDIA for computing resources. We thank Nieduszynski group members for helpful discussion and advice, and A. Carr, A. Donaldson, S. Hiraga and D. Sherratt for critical reading of the manuscript. 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.

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Contributions

C.A.N., C.A.M. and M.A.B. designed the study. C.A.M. designed and performed the experiments. M.A.B. designed and implemented the analoge training and detection software. C.A.M. and M.A.B. undertook data analysis. P.S. undertook mass spectrometry, supervised by B.M.K. and S.K. C.A.N. and J.T.S. supervised the study. C.A.N., C.A.M. and M.A.B. wrote the paper.

Corresponding authors

Correspondence to Carolin A. Müller or Conrad A. Nieduszynski.

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J.T.S. receives research funding from ONT and has received travel support to attend and speak at meetings organized by ONT.

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Supplementary Information

Supplementary Figs. 1–12 and Supplementary Tables 2 and 3

Reporting Summary

Supplementary Table 1

Information relating to D-NAscent, mass spectrometry and BrdU-seq samples.

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Müller, C.A., Boemo, M.A., Spingardi, P. et al. Capturing the dynamics of genome replication on individual ultra-long nanopore sequence reads. Nat Methods 16, 429–436 (2019). https://doi.org/10.1038/s41592-019-0394-y

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