Clonal evolution with hematopoietic differentiation. (A) Targeted resequencing of sorted CMPs, GMPs, and MEPs by NGS for UPN 6 (200 cells per fraction). (Upper) Proportion of mutated cells represents twice the VAF, determined by NGS, of heterozygous mutations in autosomes and onefold the VAF of mutations of a gene located on the X chromosome in this male subject. Bars represent 95% CIs. (Lower) Kernel density plot of VAFs determined by NGS representation facilitating inference of the presence and genetic composition of subclones (SiClone). (B) Schematic representation of the clonal architecture of CD34⁺CD38⁻ hematopoietic stem cell and progenitor compartments at diagnosis. Each mutation is represented by its initial. A color spectrum is used for the different intervals of variant allele frequencies. (C) Mutation pattern of granulocytic and erythroid precursors. Patient (UPN 6)-sorted CD34⁺ cells were expanded in vitro and transitioned to granulocytic or erythroid differentiation. Mutations were tracked by Sanger sequencing in granulocytic and erythroid differentiated cells (*point mutations). (D) Clonal evolution of UPN 6 over 20 months. World Health Organization (WHO) classification, blood and bone marrow parameters, treatments, mutations, and cytogenetic abnormality are indicated.