Separate SF3B1-mutant clones arise independently decades before clinical disease and active clonal competition. (A) Kinetics of ddPCR-assessed VAF of separate SF3B1^mt clones in BM mononuclear cells, clinical hemoglobin levels and BM ring sideroblast frequency of Patient 1, a patient with MDS-RS with separate SF3B1 N626D/K666N clones followed up over 14 years. (B-C) ddPCR–assessed SF3B1 VAFs from 3-dimensional in vitro culture time points of BM mononuclear cells seeded at (B) the inversion point of Patient 1’s long-term follow-up, and (C) the diagnostic visit of Patient 2, a patient with MDS-RS with separate SF3B1 K700E/K666N clones. (D-E) Genotype frequencies of colonies derived from sorted HSPCs and cultured in colony-forming unit assays from Patient 1 (D) and Patient 2 (E). (F-G) Clonal composition dendrograms and estimated age intervals for acquisition of each SF3B1 mutation by Patient 1 (F) and Patient 2 (G), relative to time of MDS-RS diagnosis. Error bars indicate 95% lower and upper bound errors for the age interval estimate. Comutations shared in all colonies of the same clone: P1-SF3B1^N626D: GPATCH1^N787S, EEIG2^R172S, RLBP1^K270Q; P1-SF3B1^K666N: INTS^V124F; P2-SF3B1^K666N: CPNE^R517C, PCSK1^E638K, SLC4A5^P30S, FBXO34^S320N; P2-SF3B1^K700E: TTC12^A389V, OR4N2^T259M, KAT2A^P691L. ddPCR, digital droplet polymerase chain reaction; MPD, months post diagnosis.