Fig. 2

Single-molecule detection of DNA replication dynamics on ultra-long nanopore sequencing reads. A Schematic of the experimental strategy with sequential additions of BrdU for detection of replication dynamics by DNAscent. B, C Example nanopore sequencing reads, aligned to the human genome, from HeLa-S3 cells cultured as described in Fig. 2 A. Black dots indicate BrdU probabilities at each thymidine position and the blue line indicates the fraction BrdU incorporation in independent 290 thymidine windows. Above each example read are shown: replication forks with direction (arrows), initiation (light green bars), and termination sites (dark green bars), all computationally determined from DNAscent data. Ok-seq initiation zones (dark blue bars) and ORM initiation zones (yellow bars) are also shown, from HeLa-S3 datasets. D The summary plot (upper panel) shows the mean signal across DNAscent initiation sites (± 100 kb) for proportion GC content and initiation zones from Ok-seq (HeLa-S3), Pu-seq (HCT116), and optical mapping (ORM; HeLa-S3) in blue. Also plotted are random expectations (black line) and 99% confidence intervals (gray band) from 1000 randomizations of DNAscent initiation site coordinates. The lower panel represent the same data as heatmaps (IZs in yellow) ordered by replication timing (blue) where each row is an individual DNAscent replication initiation site