Replication landscape of the S. cerevisiae genome by nanopore sequencing

Genome replication mapping methods profile cell populations, masking cell-to-cell heterogeneity. Here, we describe FORK-seq, a nanopore sequencing method to map replication of single DNA molecules at 200-nucleotide resolution. By quantifying BrdU incorporation along pulse-chased replication intermediates from S. cerevisiae, we oriented 58,651 replication tracks reproducting population-based replication directionality profiles and mapped 4,964 and 4,485 individual initiation and termination events, respectively. Although most events clustered at known origins and fork merging zones, 9% and 18% of initiation and termination events, respectively, occurred at many locations previously missed by population methods. Thus, FORK-seq reveals the full extent of cell-to-cell heterogeneity in DNA replication.