Fig. 3

Clonality and neutrality in the discovery cohort. A Clonality was assessed as described [36, 38]. Cases #1, #2, #3, #4, #8, and #9 are highly unbalanced and additional samples would be needed for correct estimation of clonality. In three cases (cases #5, #6, and #7), we could be fairly certain that mutations from the root of the phylogenetic tree were indeed clonal using the existing number of samples. B, C The neutral model assumes that there are no selective differences, such that the number of mutations of a certain allelic frequency declines as the inverse of that frequency [38]. Here, we show the agreement between each tumor sample and this neutral expectation. B Illustrates neutrality analysis of the samples from case #3. Left column: variant allele frequency histogram. Dark gray shade marks interval used for comparison with the neutral model. Central column: shows increment in the cumulative number of mutation with inverse allelic frequency 1/f (black dots) and linear model best fit (red line). Light gray marks samples that are in agreement with the neutral model R2 ≥ 0.98. Right column: normalized cumulative distribution of mutations and theoretical model. Distance between distributions was quantified using a Kolmogorov-Smirnov test. While the figure for the combined VAF shows deviations from neutrality, here mostly driven by sample G04283, some parts of the tumor could still evolve under neutral conditions. C Summarizes neutrality analyses for cases #1 to #5, #7 to #9. Case #6 (MSI) was not included in the neutrality analysis as a large, likely clonal, peak covered the most of the frequency range obfuscating the distribution of subclonal mutations. The agreement is quantified by the Kolmogorov-Smirnov test, where the Kolmogorov distance between the empirical and the theoretical distribution is shown for each sample. The normalized cumulative number of putatively subclonal mutations in a frequency area below the clonal peak was used where a power-law distributed subclonal tail of mutations would be expected in the model of neutral evolution. The lines represent the standard deviation of the Kolmogorov distance across samples per patient