Integrated analysis of leukemic mutations and transcriptomes at the single-cell level

Single-cell RNA-sequencing-based characterization of cells that belong to the neoplastic clone is a major challenge in hematologic neoplasms, where malignant and normal cells coexist. Confident molecular profiling requires simultaneous analysis of gene expression and genetic mutations in individual cells, an ability that is not supported by the standard 10X Genomics workflow. Here, we developed a post-hoc targeted genotyping method for samples processed with the 10X Genomics 3 workflow. To establish the approach, we mixed two types of leukemic cells harboring distinct mutations and subjected them to single-cell RNA-sequencing. Repurposing an intermediate product of the experimental process allowed us to enrich for transcripts containing mutation sites. Long-read PacBio sequencing genotyped the transcripts and captured the associated cellular and molecular barcodes, allowing us to bioinformatically integrate the mutation and transcriptomic data at single-cell resolution. Our method demonstrates the detection of mast cell leukemia-associated point mutations in the KIT gene and chronic myeloid leukemia-associated BCR::ABL1 fusion transcripts. Single-cell analysis of primary leukocytes from chronic myeloid leukemia detected mutated cells at diagnosis, but not during imatinib treatment. Taken together, the method constitutes a broadly applicable framework for post-hoc genotyping of cells analyzed with single-cell RNA-sequencing.