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| Status |
Public on Mar 29, 2019 |
| Title |
Capturing the dynamics of genome replication on individual ultra-long nanopore sequence reads |
| Organisms |
Saccharomyces cerevisiae; synthetic construct |
| Experiment type |
Other
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| Summary |
The replication of eukaryotic genomes is highly stochastic, making it difficult to determine the replication dynamics of individual molecules with existing methods. We now report a sequencing method for the measurement of replication fork movement on single molecules by Detecting Nucleotide Analogue signal currents on extremely long nanopore traces (D‑NAscent). Using this method, we detect BrdU incorporated by Saccharomyces cerevisiae to reveal, at a genomic scale and on single molecules, the DNA sequences replicated during a pulse labelling 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 kb, to generate the first whole genome single-molecule map of DNA replication dynamics and discover a new class of low frequency stochastic origins in budding yeast.
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| Overall design |
Direct detection of BrdU by Nanopore (ONT) sequencing
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| Contributor(s) |
Müller CA, Boemo MA, Spingardi P, Kessler BM, Kriaucionis S, Simpson JT, Nieduszynski CA |
| Citation(s) |
31011185 |
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| Submission date |
Oct 29, 2018 |
| Last update date |
Apr 24, 2019 |
| Contact name |
Conrad A Nieduszynski |
| Organization name |
Earlham Institute
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| Lab |
DNA replication & genome stability
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| Street address |
Norwich Research Park
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| City |
Norwich |
| ZIP/Postal code |
NR4 7UZ |
| Country |
United Kingdom |
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| Platforms (3) |
| GPL19756 |
Illumina NextSeq 500 (Saccharomyces cerevisiae) |
| GPL25738 |
MinION (synthetic construct) |
| GPL25739 |
MinION (Saccharomyces cerevisiae) |
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| Samples (21)
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| Relations |
| BioProject |
PRJNA499108 |
| SRA |
SRP167163 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE121941_RAW.tar |
185.0 Mb |
(http)(custom) |
TAR (of BED, FA, PSL, ZIP) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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