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<jats:title>Abstract</jats:title><jats:p>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 <jats:underline>D</jats:underline>etecting <jats:underline>N</jats:underline>ucleotide <jats:underline>A</jats:underline>nalogue <jats:underline>s</jats:underline>ignal <jats:underline>c</jats:underline>urrents on <jats:underline>e</jats:underline>xtremely long <jats:underline>n</jats:underline>anopore <jats:underline>t</jats:underline>races (D-NAscent). Using this method, we detect BrdU incorporated by <jats:italic>Saccharomyces cerevisiae</jats:italic> 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.</jats:p>

Original publication

DOI

10.1101/442814

Type

Working paper

Publisher

Cold Spring Harbor Laboratory

Publication Date

17/10/2018