704578ab-6a0e-4c8b-a6b7-44427a3b4f45
Genome-wide mapping of individual replication fork velocities using nanopore sequencing
Little is known about replication fork velocity variations along eukaryotic genomes, since reference techniques to determine fork speed either provide no sequence information or suffer from low throughput. Here we present NanoForkSpeed (NFS), a nanopore sequencing-based method to map and extract the velocity of individual replication forks detected as tracks of the thymidine analog bromodeoxyuridine incorporated during a brief pulse-labelling of asynchronously growing cells. NFS accurately retrieves previous genome-wide, mean speed estimates in Saccharomyces cerevisiae and precisely quantifies speed changes in cells exposed to hydroxyurea and in mutants with altered replisome progression. NFS surpasses standard approaches in simplicity, rapidity, throughput, spatial and temporal resolutions. Furthermore, the positioning of >125,000 single fork velocities provides the first-ever genome-wide map of fork progression based on the direct measurement of individual replication fork rates, showing a largely uniform fork speed across yeast chromosomes except for a marked slowdown at known pausing sites. BO to BY samples in the training and testing dataset correspond to various mixtures of BrdU and thymidine (0:100; 10:90; 20:80; 30:70; 40:60; 50:50; 60:40; 70:30; 80:20; 90:10 and 100:0).
Little is known about replication fork velocity variations along eukaryotic genomes, since reference techniques to determine fork speed either provide no sequence information or suffer from low throughput. Here we present NanoForkSpeed (NFS), a nanopore sequencing-based method to map and extract the velocity of individual replication forks detected as tracks of the thymidine analog bromodeoxyuridine incorporated during a brief pulse-labelling of asynchronously growing cells. NFS accurately retrieves previous genome-wide, mean speed estimates in Saccharomyces cerevisiae and precisely quantifies speed changes in cells exposed to hydroxyurea and in mutants with altered replisome progression. NFS surpasses standard approaches in simplicity, rapidity, throughput, spatial and temporal resolutions. Furthermore, the positioning of >125,000 single fork velocities provides the first-ever genome-wide map of fork progression based on the direct measurement of individual replication fork rates, showing a largely uniform fork speed across yeast chromosomes except for a marked slowdown at known pausing sites. BO to BY samples in the training and testing dataset correspond to various mixtures of BrdU and thymidine (0:100; 10:90; 20:80; 30:70; 40:60; 50:50; 60:40; 70:30; 80:20; 90:10 and 100:0).