Journal
NATURE METHODS
Volume 16, Issue 5, Pages 429-+Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/s41592-019-0394-y
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Funding
- Biotechnology and Biological Sciences Research Council [BB/N016858/1]
- Wellcome Trust [110064/Z/15/Z]
- Ontario Institute for Cancer Research through Government of Ontario
- Government of Canada through Genome Canada
- Ontario Genomics [OGI-136]
- Medical Research Council
- Ludwig Cancer Research
- Wellcome Trust [110064/Z/15/Z] Funding Source: Wellcome Trust
- BBSRC [BB/M001873/1, BB/N016858/1] Funding Source: UKRI
- MRC [1790304] Funding Source: UKRI
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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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