Extended Data Fig. 8: Validation of APOBEC3 hypermutation of ecDNA in three independent cohorts.

a, Distribution of clustered substitutions (left) and clustered indels (right) across three validation cohorts. Clustered substitutions were subclassified into DBSs, MBSs, omikli, kataegis, and other clustered mutations. Top: Each black dot represents a single cancer genome. Red bars reflect the median clustered TMB and the percentage of clustered mutations contributing to the overall TMB of a given sample for each cancer type. Middle: The proportion of each subclass of clustered events for a given cancer type with the total number of samples having at least a single clustered event over the total number of samples within a given cancer cohort. Bottom: Percentage of clustered mutations compared to the percentage of clustered driver events for substitutions (left) and indels (right). P-values were calculated using a Fisher’s exact test and corrected for multiple hypothesis testing using Benjamini–Hochberg FDR procedure. b, Left: The mutational spectrum of all kyklonas across the validation cohorts. Right: The proportion of kyklonic events attributed to SBS2 and SBS13 (p-value determined using a Z-score test; Methods). c, The proportion of samples with ecDNA that co-occur with kataegis, do not co-occur with kataegis, or do not have any detected kataegic activity across each cohort. d, YTCA versus RTCA enrichments per sample with kyklonas, where YTCA and RTCA enrichment is suggestive of higher APOBEC3A or APOBEC3B activity, respectively. The RTCA/YTCA fold enrichments were compared to the fold enrichments of non-clustered mutations (p-values calculated using a two-tailed Mann–Whitney U-test). e, The proportion of ecDNA with kyklonas that contain multiple kyklonic events. The total number of ecDNA with kyklonas are displayed above each bar plot for each cancer type.