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

Accepted version
Peer-reviewed

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Authors

Boemo, Michael A 
Spingardi, Paolo 
Kessler, Benedikt M  ORCID logo  https://orcid.org/0000-0002-8160-2446
Kriaucionis, Skirmantas 

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 .

Description

Keywords

Bromodeoxyuridine, DNA Replication, DNA, Fungal, Genome, Genome, Fungal, Genomics, High-Throughput Nucleotide Sequencing, Nanopores, Saccharomyces cerevisiae, Software

Journal Title

Nat Methods

Conference Name

Journal ISSN

1548-7091
1548-7105

Volume Title

16

Publisher

Springer Science and Business Media LLC

Rights

All rights reserved