Abstract
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 Saccharomyces cerevisiae, we orient 58,651 replication tracks reproducing population-based replication directionality profiles and map 4964 and 4485 individual initiation and termination events, respectively. Although most events cluster at known origins and fork merging zones, 9% and 18% of initiation and termination events, respectively, occur at many locations previously missed. Thus, FORK-seq reveals the full extent of cell-to-cell heterogeneity in DNA replication.
Highlights
Eukaryotic DNA replication initiates at multiple replication origins and terminates wherever converging replication forks happen to meet
We developed Python scripts to automatically detect and orient BrdU tracks and compared the resulting replication fork directionality (RFD) profiles with independent RFD profiles generated by sequencing purified Okazaki fragments [14, 22]
As previously shown [14], initiation regions detected as ascending RFD segments coincided with the position of known yeast origins. These results demonstrate that our BrdU detection methods (CNN and transition matrices (TM)) and track orientation procedures are robust and precise and that nanopore sequencing of pulse-chase-labeled BrdU replication tracks, hereafter referred to as FORK-seq, is a valid alternative to OK-seq to produce genome-wide RFD profiles
Summary
Eukaryotic DNA replication initiates at multiple replication origins and terminates wherever converging replication forks happen to meet. Understanding this process is essential as replication perturbations can threaten genome stability. DNA microarrays and massive DNA sequencing techniques have triggered an explosion of genome-wide replication mapping studies in the last decade. These cell population-based methods only provide an average profile of DNA replication where cell-to-cell heterogeneity is masked. A high-throughput single-molecule method is required to reveal this heterogeneity. DNA combing, the modern, fluorographic version of DNA fiber autoradiography, relies on antibody detection of nucleotide analogs incorporated during replication, combined with in situ hybridization of stretched DNA molecules with DNA probes, to reveal
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