To determine which mutations are present together in a single cell and to confirm that cells from the unrelated clones do not harbour any of the ancestral mutations present in the MDS clone, we performed sequencing on single-cell-derived CFU-GEMM colonies. Representative mutations are sequenced from each (sub)clone. (a) UPN08: only colonies harbouring the two mutations linked to the unrelated clone are found at this time point. The two investigated mutations from the MDS clone are absent in these colonies. (b) UPN09: most colonies only contain an EIF3L mutation corresponding to the major unrelated clone. Two colonies harbour an additional CHRM2 mutation corresponding to a descendent of the major unrelated clone. The mutations from the MDS clone are absent in these colonies. (c) UPN10: the JAK2 clone is an independent clone not containing mutations from the major MDS clone. Furthermore, this analysis confirms that LRRC34 is a descendent of the major MDS clone that later also acquired an MLL2 mutation. The mutations in FRMD8, OCA2 and PRPS1L1 never co-occur with the LRRC34 and MLL2 mutations, indicating that these are separate clones. The FRMD8 mutation appears to be a later event than the acquisition of OCA2 and PRPS1L1. The absence of a mutation (VAF <5%) is indicated in grey. The presence of a mutation (VAF >40%) is indicated with a colour that corresponds to the clones in Fig. 3.