Abstract
Balancing replication fork progression and origin usage is essential to maintain genome stability, but measuring replication fork progression rates and origin usage throughout the genome has been challenging. Here, we use nanopore sequencing combined with DNAscent to measure replication fork progression together with origin and termination site usage with single-molecule precision throughout the Drosophila genome with nearly full genome coverage. We find that replication fork progression rates are not uniform throughout the genome. Rather, fork progression is slowest in euchromatin, and this is not correlated with active transcription. Replication origins are also influenced by chromatin, but the exact position of initiation is highly variable and are often several kilobases away from ORC binding sites. Termination sites lack any chromatin or sequence motifs and appear nearly random throughout the genome. By measuring DNA replication dynamics at near full genome coverage, our work reveals key principles of metazoan replication dynamics.