Extended Data Fig. 2: Quantifying replication fork directionality.

a, Replication time profile of an example 15 Mb of C3H genome chromosome 8 (x-axis, shared with panel c). Curves show early/late (EL) replication relative enrichment (E and L read counts normalised to their respective library read depth, then relative enrichment, RE = (E − L)/(E + L)) where more positive values indicate earlier replication and more negative values indicate later replication. Replication profiles shown for a mouse embryonic stem cell line (E14TG2a, tan) and mouse hepatocyte derived cell lines (Hep-74.3a, red; Hepa1-6, brown). Blue dash line indicates the centre of a strong replication origin region (schematic) and is projected into panel c for comparison. b, Schematic illustrating two alternate strategies to generate replication fork directionality measures (RFD). Left side, E/L-Repli-seq (top) can be used to derive Repli-seq based replication fork RFD (repli-RFD; bottom). On the right side, Okazaki fragment sequencing based RFD (OK-RFD). c, Smoothed derivatives of Hep-74.3a E/L-Repli-seq data (red, panel a) provides an RFD estimate. Comparison to OK-seq data from another differentiated cell type (pink, activated B-cells) shows overall good concordance but captures some replication profile differences between cells (grey triangle). d, Kernel density plot summarising the genome-wide correlation of B-cell derived OK-RFD (x-axis) and Hep-74.3a derived repli-RFD (y-axis), both at 10 kb resolution. Only high-concordance genomic intervals between blue stepped lines (21 quantile boundaries) were used for RFD based measures of liver tumour mutation rate. e, Validation of the E/L-Repli-seq to RFD measure in human RPE-1 cells where both OK-seq (grey) and E/L-Repli-seq (black) has been generated and used to calculate RFD. The curves are shown over a 15 Mb interval of human chromosome 8 and illustrate a high concordance of RFD profile. Although both traces are plotted at 10 kb resolution, the smoothing and processing required to calculate RFD from E/L-Repli-seq averages out some of the fine grained structure evident in the OK-seq derived profile. f, Kernel density plot summarising the OK-seq (x-axis) and E/L-Repli-seq (y-axis) RFD estimates for RPE-1 cells, as for panel d.