Fig. 2

Performance of the DNAscent v2 detect subprogram. (a) When the thymidine analogue BrdU is pulsed into S-phase cells, BrdU is incorporated into the newly replicated nascent DNA in place of thymidine. Detecting BrdU in nascent DNA sequenced with ONT can reveal the movement of replication forks in millions of single molecules. (b) ROC curves showing the ratio of positive BrdU calls to false positive BrdU calls for four different experiments with different BrdU-for-thymidine substitution rates. The 26% and 49% BrdU samples are from [9] while the 38% and 69% samples are from [10]. The BrdU-for-thymidine substitution rate as measured by mass spectrometry is indicated by the dashed red line. Points along each curve are different thresholds above which a BrdU call is considered positive; for DNAscent v2, these are probabilities whereas for DNAscent v1 and below, these are log-likelihoods. Each curve was calculated using 5,000 reads. The x-axis of each plot has been truncated from 0-100% to 0-20% for clarity. Only results for DNAscent are shown, as RepNano does not call BrdU with single-nucleotide resolution. (c) Bedgraphs visualised in IGV [12] showing the proability of BrdU called at each thymidine position for a randomly selected subset of reads used in the 49% BrdU ROC curve analysis. Each track is a single read, and the y-axis of each track ranges from 0 to 1. (d) The median probability of BrdU called by DNAscent v2 at each thymidine position along primer extension reads (N=273) from [9] where BrdU has been substituted for thymidine in two known positions (30 and 36 bp) on the forward strand. All other positions on the forward and reverse strand are unsubstituted. (e)S. cerevisiae rDNA consists of 150-200 9.3 kb repeats, each of which has an origin of replication (top track) and a replication fork barrier (vertical lines) that block rightward-moving forks. There is a sharp drop-off in BrdU incorporation where rightward-moving forks hit the barrier, indicating a fork pause or stall. A total of 25 reads are shown, and for a representative read, the inset zooms in on a 1 kb region that includes the barrier