Mutational burden in matched stem and progenitor cells in the BM and differentiated cells in PB. (A) Blood differentiation hierarchy in MDS/CMML showing stem cells (healthy stem cells [HSCs/MPP] and MDS-SC), progenitors (common myeloid progenitors [CMP], granulocyte monocyte progenitors [GMP], megakaryocyte eythroid progenitors [MEP], and common lymphoid progenitors [CLP]), and differentiated mature cells (left). Those cell types colored white were not characterized in this figure. Schematic showing collection and cell sorting strategies for PB and BM (right). PB was flow sorted into neutrophils (Neut: SSC^hi, CD45⁺, immunoglobulin D–negative, CD16⁺, and CD66b⁺), monocytes (Mono: SSC^lo, CD45⁺, immunoglobulin D–negative, and CD16⁺), and nBC (SSC^lo, CD45⁺, IgD⁺, and CD27⁻). BM mononuclear cells (BM-MNC) were isolated on Ficoll and used directly for bulk capture sequencing. MACS-enriched CD34⁺ cells (BM-CD34⁺) were dropped into 384-well plates for amplicon sequencing, with indexing for CD38, CD123, CD45RA, CD90, and IL1RAP, and post hoc assignment of cell type (HSC/MPP: LIN⁻, CD34⁺, CD38^lo, CD45RA⁻, CD123⁻, IL1RAP⁻; MDS-SC: LIN⁻, CD34⁺, CD38^lo, [CD45RA⁺ or CD123⁺ or IL1RAP⁺]; CMP: LIN⁻, CD34⁺, CD38⁺, CD45RA⁻, CD123⁺; GMP: LIN⁻, CD34⁺, CD38⁺, CD45RA⁺, CD123⁺; and MEP: LIN⁻, CD34⁺, CD38⁺, CD45RA⁻, CD123⁻). (B-D) (i) VAFs determined by capture sequencing in bulk BM and PB cell types (bulk VAF) and corresponding VAFs determined by amplicon sequencing in single cells (single-cell VAF). VAFs refer to alleles: in diploid cells a VAF of 0.5 indicates that every cell carries a mutated allele. VAFs >0.5 can occur where there is loss of heterozygosity or in the case of X-linked genes in male patients where each cell carries only 1 copy of the allele. For single-cell VAFs, each allele was analyzed individually, and the bar graph indicates the number of cells analyzed for each mutation in each cell type. Bars show standard error of the mean. (ii) Single-cell haplotypes. Pie charts show the proportions of cells across the hematopoietic hierarchy carrying 0, 1, 2, 3, or 4 mutations in the specified alleles; the number of individual cells analyzed for each population are as indicated in square brackets. Patient #H198302 (B), patient #H198303 (C), and patient #H198304 (D) are shown respectively. Ery, erythrocyte; Meg, megakaryocyte.