Capturing the dynamics of genome replication on individual ultra-long nanopore sequence reads.
Simpson, Jared T
MetadataShow full item record
Müller, C. A., Boemo, M., Spingardi, P., Kessler, B. M., Kriaucionis, S., Simpson, J. T., & Nieduszynski, C. A. (2019). Capturing the dynamics of genome replication on individual ultra-long nanopore sequence reads.. Nat Methods, 16 (5)https://doi.org/10.1038/s41592-019-0394-y
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 .
External DOI: https://doi.org/10.1038/s41592-019-0394-y
This record's URL: https://www.repository.cam.ac.uk/handle/1810/301589
All rights reserved