PT  - JOURNAL ARTICLE
AU  - Carolin A Müller
AU  - Michael A Boemo
AU  - Paolo Spingardi
AU  - Benedikt M Kessler
AU  - Skirmantas Kriaucionis
AU  - Jared T Simpson
AU  - Conrad A Nieduszynski
TI  - Capturing the dynamics of genome replication on individual ultra-long nanopore sequence reads
AID  - 10.1101/442814
DP  - 2018 Jan 01
TA  - bioRxiv
PG  - 442814
4099  - http://biorxiv.org/content/early/2018/10/24/442814.short
4100  - http://biorxiv.org/content/early/2018/10/24/442814.full
AB  - 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.