(A) tSNE representation of 2,734 HSPCs from eight patients and two age-matched normal donors; the samples were processed with 3′-TARGET-seq, and 3,286 highly variable genes were used for the analysis. Cells from age-matched normal donors are colored in light gray (NORMAL). Wild-type (WT) cells from patients with MF are colored in dark gray (“WT-P”). Cells carrying mutations exclusively in JAK2 are colored in blue (“J”); those carrying mutations in JAK2 and epigenetic modifiers (TET2 and ASXL1) are colored in purple (“JE”); those carrying mutations in JAK2 and spliceosome components (SF3B1, SRSF2, and U2AF1) are colored in light green (“JS”); and those carrying mutations in JAK2, spliceosome components, and epigenetic modifiers are colored in dark green (“JSE”). The gene expression matrix was batch- and donor-corrected, and genotypes were preserved.

(B) Boxplots of representative differentially expressed genes from JAK2 only (B4GALT1), JAK2+epigenetic (PITX1), JAK2+spliceosome (ZFP36), or JAK2+spliceosome+epigenetic (PHB and ZFP36) genetic subclones. Each dot represents the expression value for each single cell; boxes represent median and quartiles, and the central line represents the median for each group. Expression frequencies are shown on the bottom of each bar for each group.

(C) Boxplot of overall Pearson’s correlation of cells from normal donors and cells from MF-patient samples; the cells are grouped per donor type (normal donor or patient sample; left panel) or by the genotype groups presented in (A) (right panel). A Kolmogorov-Smirnov test provided the significance level for each comparison (∗∗∗; p value < 0.001).

(D) tSNE representation of 1,066 WT cells from six patients and two normal donors; 3,436 highly variable genes were used. The gene expression matrix was batch-corrected, and the donor effect was preserved.

(E) tSNE projection (from the same cells as in [D]) representing relative gene expression levels from selected differentially expressed inflammation-associated genes in WT cells from patients and normal donors.

(F) Enrichment of selected pathways in the same WT cells from the same samples as in (D) and (E) from normal donors and patients. A complete list of all significant genesets tested can be found in Table S5 A.

(G) tSNE projection representing relative gene expression levels from selected differentially expressed oncogenes (FOS) and tumor suppressors (ANXA1) between the same WT cells from patients and normal donors as in (D).

(H) tSNE representation of 769 WT and JAK2-only mutant HSPCs from four patients with MF (patients IF0138, IF0155, IF0157, and IF0602); we used the top 2,000 genes as identified by the Gini index from random forest analysis.

(I) Enrichment of selected HALLMARK and STAT5A pathways from the same cells as in (H), as well as cells from normal donors (NORMAL). A complete list of all significant genesets tested can be found in Tables S5 B and S5C, and specific comparisons for subclones within patients can be found in Table S5 D.