Abstract
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 .
Highlights
Genomic methods have provided insights into DNA replication and genome stability[1,2,3]
In experiments where BrdU was limiting, we showed that D-NAscent can detect the changes in BrdU incorporation frequency to reveal the direction of replication forks and identify the location of replication origins on individual molecules
The Oxford Nanopore Technologies (ONT) MinION instrument determines a base sequence from the electrical readout produced as a single strand of DNA passes through a protein pore
Summary
Genomic methods have provided insights into DNA replication and genome stability[1,2,3]. A highthroughput single-molecule approach is needed to reveal the heterogeneity in DNA replication dynamics. DNA combing relies on antibody detection of nucleotide analogues incorporated on the nascent strand and can be used to determine the pattern of origin activation and fork progression in single molecules[5]. This approach is lowthroughput and provides limited temporal and spatial resolution: combed molecules are anonymous unless genomic positions are identified by probe hybridization, which is challenging for large metazoan genomes. In vitro systems are presently limited to small DNA molecules (replicated from a single origin) and so cannot recapitulate in vivo replication dynamics
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