Extended Data Fig. 3: Targeted amplicon sequencing of Apc.

a, Plot showing the overall sequencing coverage across all samples at various depths. n = 522 samples. b, Box plot showing the mean read depth between optically cleared and uncleared samples. n = 21 cleared tumours and 24 uncleared tumours. c, Schematic showing the pre-processing and mutation-calling pipeline. d, Schematic showing derivation of tumour spheroids used to confirm recombination of the transgenic Apc allele in both heterotypic and homotypic tumours. PCR for the floxed allele of Apc was performed on DNA derived from spheroids. Electrophoregram showing complete loss of the unrecombined band (209 bp) in monoclonal tumours and major-minor pairs. Positive controls shown on the right of the gel are from liver tissue of an Apc^het mice (two bands) and Apc^fl/fl animal without tamoxifen (one band). n = 6 monoclonal tumour spheroids and 10 major-minor pairs from 5 polyclonal tumours. e, Plot showing the number of amplicons covering each amino acid position in Apc. f, Schematic showing the influence of the clonal/subclonal structure on the resulting Apc VAF. Assuming a constant tumour fraction of 0.5, the sum of Apc VAFs is higher in situations of branching evolution (with more than one Apc mutation per tumour cell). g, h, Boxplot of Apc driver VAF (g) and Apc sequencing depth (h) for monoclonal tumours (one Apc mutation) and polyclonal tumours (two or more mutations). n = 154 monoclonal and 93 polyclonal tumours. Two-tailed Wilcoxon test in b, g and h. Details on the boxplots are provided in the Methods.