Extended Data Fig. 4: Timing of metastatic divergence.

a. Sample level divergence timing (early and late). Where both early and late divergence is seen in multiple metastasis samples of one case, the overall timing is defined as early. b. Orthogonal method to time metastatic divergence using primary ubiquitous arm-level loss of heterozygosity (LOH). Arm level LOH was significantly more likely to be fully clonal in late compared to early divergence (case level median early = 0.94, late = 1, Wilcoxon rank-sum test, p = 1.6e-5; sample level median, early = 0.92, late = 1, Wilcoxon rank-sum test, p = 4.7e-15). c. Orthogonal method timing divergence using primary clonal whole genome doubling (WGD). There is enrichment of early divergence in pre-WGD divergence (Fisher’s exact test, p = 0.0017). d. Orthogonal method to time metastatic divergence using simple absence/presence of mutations in the primary tumour, to define primary ubiquitous mutations. Early divergent tumours have a lower proportion of shared primary ubiquitous mutations (case level median early = 92.1%, late = 99.3%, Wilcoxon rank-sum test, p = 5.6e-8; sample level median, early = 90.7%, late = 99.6%, Wilcoxon rank-sum test, p = 4.20e-17). e. Examples of pre- and post-WGD divergence (CRUK0485 and CRUK0022, respectively). The red line represents the branch with WGD. f. Detected mutational signatures using sample unique mutations for each of the metastatic samples with sufficient mutations (more than 50). SBS31 and SBS35 represent the platinum mutation signatures. g. In patients treated with platinum chemotherapy and where platinum signature was detected in the metastases (9 samples), an enrichment was seen in sample-specific double base substitutions (Mann-Whitney-U test; treated and detected platinum signature vs. treated and no signature detected (25 samples), p = 2.58e-5; treated and detected platinum signature vs. untreated (181 samples), p = 1.32e-10). h. In cases where platinum signature was detected, putative metastasis-unique driver mutations were mapped to the most likely signature. Example case of CRUK0557 where mapping such mutations (PMS1, ASXL2, DOT1L, GRIN2A) revealed PMS1 to likely be platinum-driven. i. Schematic representation of the agent-based modelling approach used to investigate timing and patterns of metastatic seeding. j. Number of shared primary clonal mutations between simulated primary-metastasis pairs and the different mutations and selection rates. Additionally, the number of shared primary clonal mutations from TRACERx data is indicated. k. Kaplan-Meier analysis demonstrating no significant difference in early vs. late divergence (Log rank test, p = 0.47). l. Early divergence is associated with a higher proportion of current smokers (n early = 32, n late = 94; Fisher’s exact test, p = 0.005). The box plots represent the upper and lower quartiles (box limits), the median (centre line) and the vertical bars span the 5th to 95th percentiles. All tests were two-sided unless otherwise specified.