Extended Data Fig. 1: Confetti expression quantification and estimation of contribution of random collisions.

a, Schematic illustrating tissue preparation steps prior to confocal imaging. b, Confocal micrograph of Confetti-labelled crypts in sagittal (left) and cross (right) sections. Tissue was collected from an Apc^het animal 10 days after tamoxifen induction. c, Box plot showing the frequency of Confetti fluorophore expression in different intestinal regions assessed. n = 41 intestinal segments from 7 mice. d, Box plot of frequency of individual Confetti fluorophores. Two-tailed t-test. e, Schematic illustrating the influence of patch size on the heterotypic fraction assessment. If an adenoma arises from two contiguous crypts with two different Confetti labels, the resulting tumour is identified as heterotypic. However, if it arises from a patch of similarly coloured crypts, it is identified as homotypic despite being polyclonal in origin. f, Box plot showing the simulation results for estimating patch sizes for individual fluorophores. 1000 simulations were performed for each fluorophore. g, Bar chart showing the number of Confetti colours in heterotypic tumours. h, Bar chart depicting the relative sizes of Confetti-labelled clones in heterotypic tumours. n = 13 tumours from 5 mice. i, Violin plot comparing the diameters of homotypic and heterotypic tumours in the small and large intestines. Based on 162 homotypic and 98 heterotypic tumours. Two-tailed t-test. j, Scatter plot of the heterotypic fraction against tumour density (number of tumours per 1000 crypts). Linear regression adjusted R² −0.006. k, Representative heat map of the spatial tumour density for one intestinal segment. Tumours are marked by black dots, with the only heterotypic tumour in this segment labelled in white. l, Q-Q plot of heterotypic tumour spatial density against spatial density of all other tumours. Heterotypic tumours are not associated with a higher spatial density. m, Scatter plot showing the results of a Poisson model predicting the number of random tumour collisions for individual gut segments. The mean predicted number of collisions with the associated standard deviation is shown in red, with the green dot representing the observed number of heterotypic tumours for the segment. n = 37 samples from 10 mice. n,o, Examples of intestinal segments reconstructed following simulation of tumour initiation and growth. Random collision in o highlighted with black arrowhead. p, Q-Q plot of mean observed number of heterotypic tumours per imaged intestinal segment versus expected number of collisions in that segment based on simulation under conditions of random collision. Paired two-tailed t-test. q, Confocal micrograph of a segment of small intestine from a wildtype + ENU animal containing only one tumour. The inset shows that this tumour was heterotypic, consisting of at least two clones (uncoloured and yellow). Error bars denote s.d. (h, m). Details on the boxplots are provided in the Methods.