High throughput sequencing enables detailed analysis of S. cerevisiae DNA  replication
We have used three complementary deep sequencing approaches to characterise the temporal order of genome replication in S. cerevisiae. Each approach measures the increase in DNA copy number as a genomic region is replicated (in a large population of cells). We find that the use of deep sequencing provided high spatial resolution. For maximum temporal resolution we have measured replication dynamics at multiple time points during a synchronous S phase. To pinpoint replication origins we have synchronously released a checkpoint mutant (rad53-delta) into S phase under conditions of depleted dNTPs (200 mM hydroxyurea; HU) and measured the increase in DNA copy number after 60 minutes. Finally for rapid characterisation of replication dynamics in wild-type cells from an unperturbed cell cycle we have obtained replicating (S phase) and non-replicating (G2 phase) cells by fluorescence activated cell sorting (FACS) and subjected the DNA to copy number analysis. We have compared and contrasted these approaches and developed the tools required for interpreting the deep sequencing data. Overall design: Measurement of genome replication time for various S. cerevisiae strains. For each strain two samples were analysed: a replicating sample (from S phase) and a non-replicating sample (from G2 phase).