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Nucleic Acids Res. 2014 Jan;42(1):e3. doi: 10.1093/nar/gkt878. Epub 2013 Oct 1.

The dynamics of genome replication using deep sequencing.

Author information

1
School of Life Sciences, The University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, UK, Deep Seq, The University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, UK, Research Center for Epigenetic Disease, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, 113-0032, Japan and Institute for Complex Systems and Mathematical Biology, The University of Aberdeen, Aberdeen, AB24 3UE UK.

Abstract

Eukaryotic genomes are replicated from multiple DNA replication origins. We present complementary deep sequencing approaches to measure origin location and activity in Saccharomyces cerevisiae. Measuring the increase in DNA copy number during a synchronous S-phase allowed the precise determination of genome replication. To map origin locations, replication forks were stalled close to their initiation sites; therefore, copy number enrichment was limited to origins. Replication timing profiles were generated from asynchronous cultures using fluorescence-activated cell sorting. Applying this technique we show that the replication profiles of haploid and diploid cells are indistinguishable, indicating that both cell types use the same cohort of origins with the same activities. Finally, increasing sequencing depth allowed the direct measure of replication dynamics from an exponentially growing culture. This is the first time this approach, called marker frequency analysis, has been successfully applied to a eukaryote. These data provide a high-resolution resource and methodological framework for studying genome biology.

PMID:
24089142
PMCID:
PMC3874191
DOI:
10.1093/nar/gkt878
[Indexed for MEDLINE]
Free PMC Article
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