Extended Data Fig. 6: Variant allele frequency distributions demonstrate high rates of non-mutagenic replication over segregating lesions.

a–f, VAF distributions shown as probability density functions (total area under curve = 1) for six example tumours, calculated taking into account observed multiallelic variation. The VAF for identified driver mutations is indicated (brown triangle). Tumour identifiers are shown top right along with the percent of genomic segments (based on mutation asymmetry segmentation) that are multiallelic. Skew shows Pearson’s median skewness coefficient for the VAF distributions. a–c, Tumours with no multiallelic segments and exhibit a symmetric VAF distribution showing minimal sub-clonal structure. d–f, Tumours with all segments multiallelic, illustrating the sub-clonal structure generated by segregating lesions. g, Tumours with a high proportion of multiallelic segments have a left-skewed VAF distribution indicating frequent non-mutagenic replication over segregating lesions. Percent of genome segments that are multiallelic (x-axis) plotted against VAF distribution skew for 371 C3H tumours. Tumours with low estimated cellularity indicated in pink and excluded from correlation analysis (n = 335 independent tumour samples in Pearson’s correlation, two-sided significance from Fisher’s transform). h, As for g, but showing 84 CAST tumours (n = 77 independent tumours included in Pearson’s correlation). i, Mutation asymmetry summary ribbon for example C3H tumour 90797_N2; C3H genome on the x-axis. The percent of mutation sites with robust support for multiallelic variation (y-axis) calculated in 10Mb windows (grey) and for each asymmetric segment (black). Thresholds for high (black), intermediate (grey) and zero (red) rates of multiallelic sites shown on the right axis. j, VAF density plots for the example tumour 90797_N2 (shown in i) mutations in asymmetry segments stratified by the multiallelic rate thresholds defined in i. As with individual tumour based analysis (a–h), high multiallelic rates correspond to a leftward skew of the VAF (black, grey) whereas segments without multiallelic variation (red) show a minimally skewed distribution.