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Telomere-to-telomere DNA replication timing profiling using single-molecule sequencing with Nanotiming.
Nat Commun, 16(1):242.
Current temporal studies of DNA replication are either low-resolution or requirecomplex cell synchronisation and/or sorting procedures. Here we introduceNanotiming, a single-molecule, nanopore sequencing-based method producinghigh-resolution, telomere-to-telomere replication timing (RT) profiles ofeukaryotic genomes by interrogating changes in intracellular dTTP concentrationduring S phase through competition with its analogue bromodeoxyuridinetriphosphate (BrdUTP) for incorporation into replicating DNA. This solely demandsthe labelling of asynchronously growing cells with an innocuous dose of BrdUduring one doubling time followed by BrdU quantification along nanopore reads. Wedemonstrate in S. cerevisiae model eukaryote that Nanotiming reproduces RTprofiles generated by reference methods both in wild-type and mutant cellsinactivated for known RT determinants. Nanotiming is simple, accurate,inexpensive, amenable to large-scale analyses, and has the unique ability toaccess RT of individual telomeres, revealing that Rif1 iconic telomere regulatorselectively delays replication of telomeres associated with specific subtelomericelements.
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