High-throughput analysis of DNA replication in single human cells reveals the complex nature of replication timing control

Abstract

AbstractDNA replication initiates from replication origins firing at different times throughout S phase. 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. We developed an approach to profile DNA replication from whole-genome sequencing of thousands of single cells. We describe “in silico flow cytometry”, a method for discriminating replicating cells with superior accuracy to FACS and staging them across S phase. Using two microfluidic platforms, we analyzed up to 2,428 individual replicating cells from a single sample. The resolution and scale of the data allow focused analysis of replication initiation sites, demonstrating that the vast majority are in confined genomic regions. While initiation occurs in a remarkably similar order across cells, we unexpectedly identified a subset of initiation regions that constitutively fire in late S phase, and another subset firing randomly throughout S phase. Taken together, high throughput, high resolution sequencing of individual cells reveals previously underappreciated variability in replication initiation and progression.