Fig. 3

a–c Single-cell multiplex qPCR evolutionary analysis samples 6030, 6116 and CUL76. Evolutionary trees generated by bioinformatics analysis of single-cell data are shown. Gene names of drivers tracked are shown next to the subclones. Presence of a gene name indicates gene mutation present in the case of SNVs or indels. Where the driver is a deletion number of copies of gene present (0 or 1) is shown. Yellow circles represent leukaemic subclones and a black circle and the N label indicates the normal state. The size of the circle is proportional to the number of cells in each subclone and the detected genetic markers are listed below each circle. Red T = subclones that read-out in xenotransplants (detailed xenograft single-cell data in Supplementary Table 16). Grey boxes represent inferred subclones; these are groups of cells, which have died out, been outcompeted or if still present, exist at low frequencies below the level of reliable detection using this approach. Tree branch lengths are directly proportional to the number of evolutionary changes inferred and the points at which the branches diverge (nodes) represent the ancestor state of a clonal clade; a monophyletic group, which includes all descendants of the ancestor. The number in grey colour at each node indicates the bootstrap value. The phylogeny shows how the clonal expansion has evolved from a common ancestor toward the observed states. Note that for case CUL76 as a limited number of drivers were tracked based on mutation screening and copy number data another known mutation present in this sample (SORC3, passenger mutation) was included to aid assessment of clonal structure. a Case 6116 (N = 308 cells). The root of the tree harbours the STIL-TAL1 fusion (F) and a homozygous CDKN2A deletion. Two equally parsimonious trees (I and II) are generated which differ as to whether the FREM2 or PIK3CD mutated clone gives rise to the latest detectable C4 clone. The NOTCH1 mutation is a subclonal event. b CUL76 (N = 151 cells). The root of the tree harbours the STIL-TAL1 F and a homozygous CDKN2A deletion and a LMO2 deletion in this case. PTEN inactivation, through copy number loss, and NOTCH1 mutation are subclonal. The NOTCH1 TAD domain exon 34 mutation was only detected in clone C2 (NOTCH1 mutation status determined by single-cell Sanger sequencing of 44 single cells). The pie chart shows the proportions of C2 subclone cells that are either wild-type (WT) or NOTCH1 mutated. c 6030 (N = 216 cells). The root of the tree harbours the STIL-TAL1 F and a homozygous CDKN2A deletion. PTEN mutations are subclonal and re-iterative (3 PTEN indels in exon 7—labelled PTEN indels 1, 2 and 3 plus one PTEN indel in exon 8). The C6 subclone contains two independent PTEN exon 7 mutations. PTEN exon 8 mutation was only detected in clone C7 (PTEN exon 8 mutation status determined by single-cell Sanger sequencing of 37 single cells). The pie chart shows the proportions of C7 subclone cells that are WT, heterozygous PTEN exon mutated or homozygous PTEN exon 8 mutated