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Furthermore, some recent replication origin-mapping methods have indicated that replication origins are highly abundant and highly dispersed throughout the human genome 1, 7, suggesting that many sites may function as origins used in a subset of cell cycles. Ensemble replication-timing measurements have been interpreted to indicate that replication is organized in broad domains, spanning hundreds of kilobases to several megabases, with consistent replication timing governed by the activity of clusters of replication origins 5, 6.
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Furthermore, debate persists over whether the reproducible nature of the replication-timing program reflects the consistent activity across cells of specific individual replication origins or stochastic firing of different origins in different cells within a given region. However, the molecular mechanisms that determine the locations and preferred activation times of replication origins in mammalian genomes remain unclear. This replication-timing program is highly reproducible across experiments 2, suggesting strict regulatory control and conserved across phylogeny 3, 4, suggesting selection under evolutionary constraint. In many eukaryotes, sequencing of cells at different stages of the cell cycle has been used to profile DNA replication timing, which measures the relative time that different genomic regions are replicated during S phase (reviewed in 1). Eukaryotic DNA replication initiates at replication origin loci, which are licensed in the G 1 phase of the cell cycle and fired at different times during the S phase. Taken together, high throughput, high resolution sequencing of individual cells reveals previously underappreciated variability in replication initiation and progression.įaithful duplication of the genome is a critical prerequisite to successful cell division. While initiation order is remarkably similar across cells, we unexpectedly identify several subtypes of initiation regions in late-replicating regions. The resolution and scale of the data allow focused analysis of replication initiation sites, demonstrating that most occur in confined genomic regions. Using two microfluidic platforms, we analyze up to 2437 replicating cells from a single sample. We develop an approach to profile DNA replication from whole-genome sequencing of thousands of single cells, which includes in silico flow cytometry, a method for discriminating replicating and non-replicating cells. Debate remains about whether origins are a fixed set of loci, or a loose agglomeration of potential sites used stochastically in individual cells, and about how consistent their firing time is.
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DNA replication initiates from replication origins firing throughout S phase.