Differential expression and Gene Ontology enrichment analysis (GOEA).

(A) Total number of inferred replication cycle differentially expressed genes (DEGs) in each species independent of other species. (B) Stack bar plot showing proportion of DEGs in each phase in all species. (C) Bar plot showing the total number of conserved DEGs of inferred replication cycle phases (rows) across all species. (D) Total number of DEGs of the indicated phase unique to the indicated species (colors). The most significant Gene Ontology (GO) term (Benjamini 2 and adjusted p-value S3 and S4 Tables. Data and code for generating the figure are available at https://github.com/umbibio/scBabesiaAtlases.</p

Additional figures.

S1 Text also contains extended analysis on species-specific markers and the human versus bovine marker analysis. (PDF)</p

Gene clusters.

Tabs 1 and 2 contain the list of genes in each cluster for epigenetic markers (Fig 8) and TFs and AP2s (Fig J in S1 Text). (XLSX)</p

Functionally related genes.

Tabs 1–3 contain the list of orthologous Babesia transcription factors (TFs), AP2s, and epigenetic markers. Tabs 4–6 contain the list of VESA genes in B. bovis, B. bigemina, and B. divergens. (XLSX)</p

Host-specific differential expression and GO term enrichment analysis.

(A) Top: Expression of top differentially expressed genes in B. divergens in bovine and human RBCs. Violin plots illustrate the distribution of expression of the top DEGs. Bottom: Bar plots showing the total number of DEGs in the indicated phase (rows) in each sample (colors). (B) Top: Expression of top DEGs in combined B. bigemina, B. bovis, and B. divergens in bovine RBCs versus B. divergens in human RBCs. Violin plots illustrate the distribution of expression of top DEGs. Bottom: Bar plots showing the total number of DEGs in the indicated phase (rows) in each sample (colors). To eliminate the confounding effect caused by species differences, any DEG between B. divergens and B. bigemina or between B. divergens and B. bovis in bovine RBCs was excluded from this analysis. The topmost significant GO term associated with each set of DEGs (Benjamini https://github.com/umbibio/scBabesiaAtlases.</p

Transcription profile of replication cycle regulated genes: Heatmap of mean expression curves of differentially expressed genes of inferred replication cycle phases.

Rows represent genes and columns represent pseudo-time-ordered cells. Horizontal facets group genes in the indicated phase. Vertical facets mark the transition time points. Analysis is done in each species independently (Fig 4A). Rows are ordered according to peak expression time. Data and code for generating the figure are available at https://github.com/umbibio/scBabesiaAtlases.</p

GO terms.

Tabs 1–3 contain the list of enriched GO terms for conserved and species-specific DEGs and human versus bovine DEGs. (XLSX)</p

Quantification of alignment of gene curves between synchronized bulk and single-cell sequencing.

(A) Dynamic time warping alignment of a sample gene: high alignments (left), low alignments (right). Gray dashed lines indicate the matched time index (warping). (B) Top left: Correlation between calculated peak times in S/M/C phases, excluding the boundary. Top right: Percent alignment distance categorized into high (distance upper 80th percentile), and mid. Curves with no peak contain a higher percentage of poor alignment. Bottom: Distribution of dynamic time warping (dtw) alignment distance between single-cell and bulk sequencing. Shaded areas show boundaries of alignment categories. NA, not available. Data and code for generating the figure are available at https://github.com/umbibio/scBabesiaAtlases.</p

Projection of single-cell RNA sequencing data onto UMAP coordinates and the first 3 PC coordinates.

B. divergens in human and bovine host cells cluster together. PCA, principal component; UMAP, Uniform Manifold Approximation and Projection. Data and code for generating the figure are available at https://github.com/umbibio/scBabesiaAtlases.</p

Quality control.

Tab 1 contains the alignment metrics. Tab 2 summarizes the total number of genes and cells that pass the Seurat filters. Tab 3 summarizes the total number of significant genes in the fitted generalized additive model (GAM; adjusted p-value (XLSX)</p