a, Illustration of a portion of the B cell receptor (IGH) region on chromosome 14. Shown are the coverage tracks of an LCM sample that does not belong to the lymphocyte lineage (top) and a sample that belongs to the lymphocyte lineage (middle). In the centre of the displayed region there is a drop of copy number in the lymphocyte track, which indicates a structural rearrangement. The bottom track shows the paired-end reads that contribute to a rearrangement event in the lymphocyte sample, colocalized with the drop in copy number. b, Application of the pigeonhole principle: if two clusters of heterozygous mutations in regions of diploid copy number are in different cells, then their median VAFs must sum to ≤0.5 (if they sum to >0.5, equivalent to a combined cellular fraction of >1, then there must be some cells that carry both sets of mutations—hence one cluster would have a subclonal relationship with the other). Cluster 10 is the cluster with the unique VDJ rearrangement of IGH that is shown in a and the large number of mutations attributed to signature 9. Clearly, samples from clusters 2, 11, 55 and so on have VAFs which, when combined with cluster 10, sum to >0.5. Therefore, they must be subclonal to cluster 10, even though they do show signature 9. c–h, Representative pairwise decision graphs for clusters of mutations. The median cellular fraction is shown for pairs of clusters across every sample from the patient. Where at least one sample falls above or to the right of the x + y = 1 diagonal line, those two clusters must share a nested clonal–subclonal relationship.