TY - JOUR
T1 - Capturing the dynamics of genome replication on individual ultra-long nanopore sequence reads
AU - Müller, Carolin A.
AU - Boemo, Michael A.
AU - Spingardi, Paolo
AU - Kessler, Benedikt M.
AU - Kriaucionis, Skirmantas
AU - Simpson, Jared T.
AU - Nieduszynski, Conrad A.
N1 - 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.
PY - 2019/5
Y1 - 2019/5
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85064759461&partnerID=8YFLogxK
U2 - 10.1038/s41592-019-0394-y
DO - 10.1038/s41592-019-0394-y
M3 - Article
C2 - 31011185
AN - SCOPUS:85064759461
VL - 16
SP - 429
EP - 436
JO - Nature Methods
JF - Nature Methods
SN - 1548-7091
IS - 5
ER -