Extended Data Fig. 6: Single-cell genotyping of bone marrow samples.

a, Scatterplot shows overview of the genotyping efficiency of all 40 mutations targeted by XV-seq across 11 samples. Some mutations were analysed in multiple samples collected from the same patient. b, Genome plot shows combined 10x scRNA-seq reads for Patient 10 relapse bone marrow donor cells and host cells over the CDKN2A gene locus on chromosome 9. The focal homozygous deletion observed in (malignant) host cells results in atypical splicing of the upstream MTAP gene to five different acceptor sites downstream of CDKN2A. This enabled the generation of a genotyping primer specific to exon 4 of MTAP that can be used to detect the CDKN2A deletion in single cells. c, Scatterplot compares the number of genotyped cells detected in raw scRNA-seq data (x-axis) with the number of genotyped cells detected by XV-seq (y-axis, r = 0.71). Median enrichment across targets is 11.1-fold (indicated by dashed line). These data demonstrate XV-seq target enrichment and the utility of selecting suitable mutations based on raw scRNA-seq data. d, Scatterplot shows the genotyping efficiency of XV-seq targets (y-axis) compared to the normalized expression level of the transcript (x-axis, r = 0.55). e, Scatterplot shows agreement between VAFs from bulk targeted sequencing using the Rapid Haem Panel (y-axis) and single-cell genotyping using XV-seq (x-axis, r = 0.73). For the latter, the VAF was calculated as the number of mutated transcripts / number of total transcripts captured. f, Barplot shows the percentage of cells from the Patient 10 relapse sample (post stem cell transplant) for which genotype information was obtained (Extended Data Fig. 5e–g). RAB9A is located on chromosome X (male patient) and CDKN2A is located on chromosome 9 of which one copy is lost in addition to the focal deletion of the locus (Extended Data Fig. 3a). The exclusive detection of wild-type and mutated transcripts in the expected cell populations supports accuracy of cell type annotation, host/donor classification, and XV-seq mutation detection. g, Illustration of supporting evidence for subclonal structure obtained from single-cell XV-seq of the Patient 10 uninvolved bone marrow. TET2 mutations S792* and Q1034* co-occur in the same cell (major subclone). Similarly, TET2 mutations H1216* and H1380Y also co-occur in the same cell (minor subclone). Mutations specific to the two subclones were not detected in the same cell. The sample is karyotypically normal, further supporting the existence of two subclones, as truncating mutations in TET2 are unlikely to affect the same allele. VAFs from targeted sequencing are indicated between parentheses. h, Illustration of supporting evidence for subclonal structure obtained from single-cell XV-seq of the Patient 9 uninvolved bone marrow. This sample is characterized by a sub-clonal loss of heterozygosity (LOH) on chr7q. Detection of the lost haplotype in n=25 single cells indicates that mutations in TET2 and CUX1 occurred before the LOH of chr7q. VAFs from targeted sequencing are indicated between parentheses. The high VAF of the mutation in CUX1 is explained by its ___location on chr7q. i, Heatmaps show proportion of TET2-mutated cells in each major hematopoietic cluster. Ten patient-specific TET2 mutations were assessed in five marrow samples. P-values indicate mutant-cell enrichment in HSPC/Erythroid/Myeloid vs. B/T/NK cells by Pearson’s Chi-square test with Yates’ correction. Related to Fig. 2.