Extended Data Fig. 4: Supporting information for clonal and functional heterogeneity in malignant populations revealed by mtDNA mutations.

(a) Flow cytometry gating strategy of CLL patient derived PBMCs showing expansion of CD19 + cells. (b) Identification of high-confidence variants for Patient 1 (top) and Patient 2 (bottom). The number of variants n is indicated. (c) Inference of subclonal structure from somatic mtDNA mutations for patient 2. Cells (columns) are clustered based on mitochondrial genotypes (rows). Colors at the top of the heatmap represent clusters or putative subclones. Color bar, heteroplasmy (% allele frequency). (d) Dot plots showing the mitochondrial genome coverage (log10; y-axis) for the top 500 cells per technology for four indicated scRNA-seq technologies. (e) The mean per-position mitochondrial genome coverage for the same 500 cells as in (d). (f) Volcano plot showing differential gene expression analysis from major and minor clonotypes defined by BCR sequence. Immunoglobulin (IG) genes are shown in purple; all other genes with an FDR < 0.05 are shown in blue. (g) Results for per-peak chi-squared association with sub-clonal group. Each dot is a peak rank-sorted by the chi-squared statistic. (h) Heteroplasmy from the sum of single-cells in the CD19 + and CD19- mtscATAC-seq experiments for indicated mutations and patients. (i) Histograms showing the distribution of heteroplasmy across the profiled population of cells for six selected variants, four from Patient 1 (left) and two from Patient 2 (right). The number of variants in the top heteroplasmy bin (>90%) are shown in red. (j) Allele frequency from the sum of single cells from the 5’ CD19 + and CD19- scRNA-seq libraries for two indicated variants - chr4:109,084,804A > C (‘LEF1’) and chr19:36,394,730G > A (‘HSCT’). (k) Corroboration of T cells based on gene expression signatures and carrying indicated somatic nuclear and mtDNA mutations (Patient 2). (l) Gene activity scores supporting cell type annotations in Fig. 4n. Arrows: cluster enriched for respective gene score. (m) All mtDNA mutations (rows) by cells (columns) observed in the CRC tumor. Columns are colored by defined chromatin cell state defined as in Fig. 4n. (n,o) Chromatin-derived UMAP with cells marked by select mtDNA mutations enriched in (n) epithelial and (o) immune cells. Color bar: heteroplasmy (% allele frequency).