The barcodes were then used to reassemble the genetic profiles of cells from next-generation sequencing data. We applied this approach to sequential clinical MDS samples, genotyping the most clinically relevant loci across more than 15,000 individual cells. Additionally, to study effects of subclonal mutations on drug sensitivity, ex vivo functional testing was performed on red blood cell-lysed peripheral blood and/or bone marrow aspirate patient samples. Targeted single-cell sequencing was able to recapitulate bulk sequencing data from both peripheral blood and bone marrow aspirate samples. Furthermore, the single-cell nature of our approach enabled definitive determination of mutational co-occurrence within the same cell. For examples in one sample, bulk sequencing identified mutations in JAK2 and NRAS both at 3% variant allele frequency (VAF), where single-cell analysis suggested that these mutations were mutually exclusive, each defining a distinct subclone. Single-cell sequencing allowed for serial monitoring of clonal evolution, with analysis of sequential samples from this same patient showing increase of NRAS clone from 2.5% to 24.7% at time of disease progression after hypomethylating agent therapy. Furthermore, single-cell analysis was able to identify a distinct subclone characterized by a KRAS mutation (0.4% at initiation of therapy and 6.7% at relapse), missed by serial bulk sequencing.