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Capturing the dynamics of genome replication on individual ultra-long nanopore sequence reads

Publication

Date: 22nd April 2019 | Source: Nature Methods

Authors: Carolin A Mueller, Michael A Boemo, Paolo Spingardi, Benedikt Kessler, Skirmantas Kriaucionis, Jared T Simpson, Conrad A Nieduszynski.

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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