(a) UMAP colored by detected clusters based on gene expression profiles, (b) Heatmap of the top 10 gene markers upregulated in each of the 3 cell populations identified (log2FC > 1 and adjusted p-value < 0.05), (c) Expression of a cluster 0 marker gene (C4B63-16g183) on the UMAP, and (d) Expression of a cluster 1 marker gene (C4B63-16g155) on the UMAP.

(a) Summatory of expression levels values from all genes belonging to the disruptive compartment for each cell from amastigote (Cluster 1) and trypomastigote (Cluster 0) cell populations, (b) UMAP visualization of the expression patterns of multigene family genes. (c) Violin plots showing the number of cells expressing a specific gene belonging to each group of genes: subsampled single-copy and multigene families, ribosomal genes and trans-sialidases. To avoid biases against size differences between compartments, we generated a subsampled single-copy genes list, randomly selecting an equal number of genes as those from the multigene family’s gene set. The expression distribution of the subsampled single-copy genes is similar to the distribution of the entire dataset. (d) Boxplots depict the Gini index, across single-copy and multigene families, ribosomal genes and trans-sialidases. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. For (c) and (d), all comparisons of the multigene families against single-copy and ribosomal genes are statistically significant.

Trypomastigote sub-populations identified based on trans-sialidase expression profiles. (a) violin plot displaying average expression levels of ribosomal protein-coding genes across sub-populations. (b) violin plot showing combined trans-sialidase expression levels for each sub-population. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

(a) Heatmap displaying the expression of TcS genes in each cell that together account for 75% of total TcS gene expression within cluster trypo 0. Cells are clustered by TcS expression profiles, with colors representing each gene’s percentage contribution to the cell’s total TcS expression. (b) Average expression of TcS genes grouped by the percentage of cells expressing each gene, (c) Gini index distribution for trypomastigotes cluster 0 cells considering only TcS detected in each cell. (d) Cumulative plot showing the percentage of TcS expression in each cell from cluster trypo_0. Genes are ordered by descending expression level and sequentially added to obtain each value on the Y-axis. The observed linear increase suggests a relatively equal contribution of each gene to the overall expression.

Gene expression in amastigote and trypomastigote cell populations. To minimize biases related to size differences between compartments, we generated a subsampled single-copy genes list, by randomly selecting an equal number of genes to match those from the multigene family’s gene set. (a) For each gene set, we calculated the summatory expression of those detected genes for each cell (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001), (b) UMAP projection for 2D visualization of core gene expression among cells, (c) Violin plots showing the number of cells expressing a specific gene belonging to each group of genes: subsampled single-copy and multigene families, ribosomal genes and different multigene families, and (d) Gini-indexes for subsampled single-copy genes, multigene family genes (together or grouped by multigene families) and ribosomal protein-coding genes as a constitutive expression control group, in trypomastigotes. All comparisons among multigene families and single-copy and ribosomal genes yield statistically significant differences.

Trypomastigote sub-clusters identified based on trans-sialidase expression profiles. Violin plots displaying average expression levels across sub-clusters and associated fold changes (FCtrypo_0/trypo_1) of (a) transporters coding genes, (b) DNA and RNA polymerase-associated protein coding genes, (c) phosphatases coding genes and (d) multigene family genes. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

(a) Venn diagram showing the overlap between the top 100 most expressed TcS from bulk RNA-seq data and TcS expressed in more than 40% of cells from cluster trypo_0, and (b) Heatmap displaying the expression of TcS genes in each cell that together account for 75% of total TcS gene expression and are expressed in more than 40% of cells within cluster trypo_0. Cells are clustered by TcS expression profiles, with colors representing each gene’s percentage contribution to the cell’s expression.