FORK-seq: replication landscape of theSaccharomyces cerevisiaegenome by nanopore sequencing

Abstract

Genome replication mapping methods profile cell populations, masking cell-tocell 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 populationbased replication directionality profiles and map 4,964 and 4,485 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.

Genome-Wide Mapping of Human DNA Replication by Optical Replication Mapping Supports a Stochastic Model of Eukaryotic Replication

“…In addition to mapping replication initiation sites, we also used ORM to map ongoing replication forks in unperturbed, asynchronous cells. We inferred fork direction from the asymmetric nature of incorporation tracks caused by the reduction of incorporation as labeled nucleotides are depleted, an approach that has been used before for both radio-and fluorescently labeled nucleotides (Müller et al, 2019;Huberman and Riggs, 1968;Hennion et al, 2020). Averaged over our nearly-500-fold fiber coverage of the genome, our inferred fork directions allow us to infer genome-wide replication kinetics that agree with independently determined replication-timing and replication-fork-directionality profiles ( Figures 2C,D).…”

“…diminishes, a strategy that has been used in other fiber-analysis approaches (Müller et al, 2019;Huberman and Riggs, 1968;Hennion et al, 2020). We calculated a fork direction index (FDI) as the ratio of integrated fluorescent signal in the left half of the track to that in the right half, for all tracks with 3 or more signals.…”

“…However, to date, single-molecule replication-mapping technologies, such as DNA combing and SMARD, which have throughput of a few hundred fibers, have provided insufficient depth of coverage for metazoan genomewide analyses. Recently, nanopore sequencing has been used to map replication incorporation tracks genome-wide on single DNA fibers, but the current technology only allows for relatively small datasets of relatively short fibers (Georgieva et al, 2019;Müller et al, 2019;Hennion et al, 2020). The largest dataset from a recent study describing the DNAscent approach to nanopore-based replication mapping in budding yeast was 3.8 Gb (the equivalent of just over 1x coverage of the human genome) with an average fiber length of 32 kb (Müller et al, 2019).…”

“…However, these techniques are restricted to the analysis of at most a few genomic loci. Nanopore-sequencing-based methods offer the potential to map replication genome-wide at high resolution (Müller et al, 2019;Georgieva et al, 2019;Hennion et al, 2020), but the current throughput of nanopore sequencing does not allow genome-wide analysis of metazoan genomes and current average read lengths are on the order of only 30 kb. Optical mapping technologies provide ample throughput on individual long (150 kb to 2 Mb) DNA fibers (Lam et al, 2012) and can be combined with nucleotide analog incorporation to map DNA replication (De Carli et al, 2018).…”

An evolutionary model identifies the main selective pressures for the evolution of genome-replication profiles

“…Indeed, the genome-wide replication kinetics of a population of cells can be accessed experimentally by different techniques [9][10][11]. Recent techniques also allow to measure replication progression at the single-cell level [12,13]. The estimation of key origin parameters from data requires minimal mathematical models describing stochastic origin initiation and fork progression [10,[14][15][16].…”