Reconstructing the timeline of clonal evolution in myeloproliferative neoplasms. Schematic drawing of the timeline (not to scale) and the different hematopoietic compartments during the clonal evolution of MPNs caused by JAK2-p.V617F. Starting in embryogenesis with the first division of an ancestral HSC, the daughter cells acquire a number of mutations in their genomes that can be used as markers to distinguish them from all other cells that underwent cell division. New mutations are added during each of the next cell divisions, and by comparing the sequence similarities and differences of individual HSCs, a phylogenetic tree can be reconstructed. This analysis relies on taking bone marrow cells after MPN has been diagnosed and depositing single HSCs or progenitor cells into wells where they can be expanded in liquid culture to obtain single cell–derived colonies. DNA from each of these colonies is then analyzed by WGS. By comparing the sequence similarities and differences of individual HSCs, a phylogenetic tree can be reconstructed that originates in a common ancestor HSC that first divided at the time of gastrulation. The estimate of the time when JAK2-p.V617F mutation was acquired is calculated by assuming a constant mutational rate of 19 mutations per HSC per year. This estimate is not confounded by the increased cell division rate of JAK2-p.V617F mutant HSCs, because only sequence alterations that occurred before JAK2-p.V617F was acquired were used for deriving the estimate.