a, t-SNE visualization of the entire single-cell dataset (n = 8,924 cells). WNT (blue), SHH (red), Group 3 (yellow) and Group 4 (green) patient samples are indicated. PDX models are shown in pink. Non-neoplastic oligodendrocytes and immune cells are included for comparison. Generally malignant cells are expected to cluster by patient sample, whereas non-malignant cells are expected to cluster by cell type. Only few cells from different samples cluster with oligodendrocytes (n = 22) or immune cells (n = 6) and were classified as non-malignant. No additional clusters of cells from different samples were identified, indicating the absence of additional non-malignant cell populations in our dataset. b, Identical t-SNE visualization as in a, coloured by copy-number state. CNVs were detected in most single cells, facilitating their classification as malignant. A small number of cells did not show CNVs, even though CNVs were detected in the majority of cells from the respective sample (n = 38). These cells were classified as non-malignant. Most cells with without CNVs clustered with normal oligodendrocytes (n = 21), supporting their initial classification as non-malignant. Remaining cells without CNVs did not form clusters and likely represent poor-quality cells. c, Identical t-SNE visualization as in a, coloured by detected mutant and wild-type transcripts. Cells classified as non-malignant are depleted for mutant transcripts (P < 0.01, binomial test), supporting their initial classification. d, Heat map shows detected mutant and wild-type transcripts for 39 variants (columns) in each cell (n = 1,780, rows) of the WNT MB dataset. If both mutant and wild-type transcripts are detected in a single cell, only the mutant transcript is shown. Variants were initially detected by genome sequencing and subsequently quantified in the scRNA-seq data. Sample BCH807 was not subjected to genome sequencing, and the CTNNB1 variant was manually detected by examining scRNA-seq alignments. Mutations are detected almost exclusively in single cells from samples in which they were detected by genome sequencing, illustrating the high specificity of single-cell variant detection. e, Heat map shows mutant and wild-type transcripts for 15 variants in each cell (n = 1,135, rows) of the SHH MB dataset. Sample SJ454 was not subjected to genome sequencing, and the TP53 mutation was manually identified by examining scRNA-seq alignments. f, Heat map shows mutant and wild-type transcripts for 28 variants in each cell (n = 3,172, rows) of the Group 3/4 MB samples that were subjected to genome sequencing.