Unpicking human DNA replication with single molecules

Rosemary’s lab (at the Sir William Dunn School of Pathology, University of Oxford) is interested in the dynamics of DNA replication, such as the genomic location of the origin of replication, and how this varies between individual cells and different phases of the cell cycle, as well as aberrant changes that occur in diseases such as cancer (e.g. altered origins, or fork stalling). However, because current sequencing methods are largely population-based, individual cell heterogeneity and rare events are masked. To address this Rosemary ‘turned to single molecules, using Oxford Nanopore sequencing’, and her team developed the D-NAscent method in yeast to look at replication dynamics on single molecules by measuring BrdU incorporation (Müller, Boemo et al. [2019] Nat. Methods).

Rosemary is using the D-NAscent method of BrdU incorporation to investigate replication dynamics in human cells (HeLa cells). To obtain sufficient coverage she performed the nCATS Cas9-based enrichment protocol, followed by MinION sequencing (1 library per flow cell). Her analysis pipeline included minimap2 alignment, and DNAscent2.0 for BrdU calling. DNAscent outputs the probability of a thymidine position being a BrdU substitution.

Rosemary displayed example nascent and parental reads, containing high (60% substitution) and low (indistinguishable from baseline) BrdU read calls, respectively. She showed replication differences between specific loci: the TOP1 locus, which is known to replicate early, shows high BrdU incorporation during S-phase, contrary to a region of chromosome 7, known to replicate late. She demonstrated how this method can also detect rare events, revealing what may be important biological heterogeneity in replication in small numbers of cells. And finally, Rosemary stated how using Cas9-targeted sequencing (nCATS), regions could be enriched up to 267-fold; this could allow her to further interrogate regions of interest.