Figure 1 Adapting Seurat for high-dimensional flow cytometric data analysis retrieved robust results on adult blood (AB) peripheral blood mononuclear cells (PBMCs), confirmed by Spectre and manual analysis
A. An overview of the adult blood (AB) study design. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples from healthy adult and then were first labelled with anti-CD45 antibodies with different fluorophores or their combinations for barcoding. After that, PBMCs were pooled together and stained with our 20-marker antibody panel and analyzed with Spectral Cytometry. Next, the resulting data were first demultiplexed based on their CD45 marker signals and then subject to analysis with Seurat, Spectre, and manual gating.
B. Uniform manifold approximation and projection (UMAP) plots visualizing the clustering results from Seurat (left) and Spectre (right) based on the adult PBMC CD8+ T cell experiment. One color represents one cluster.
C. Dot plots visualizing the clusters identified by Seurat (left) and Spectre (right) and their marker expression profiles. The size of the dot corresponds to the percentage of cells expressing the corresponding markers and the color gradient reflects the average normalized expression of the corresponding markers.
D. Projection of 14 clusters identified by Seurat based on the adult PBMC CD8+ T cell experiment onto the two-dimensional (2D) plot comparing their expression of CD27 and CD45RA. The dashed lines denote the average normalized expression of CD27 and CD45RA for all cells.
E. Venn diagram comparing the clustering results from Seurat and Spectre. Both methods were set to generate 14 clusters and 11 out of 14 clusters could be identified by both methods, while Se1, Se8, and Se10 could only be identified by Seurat and Sp1, Sp8 and Sp10 could only be identified by Spectre.
F. Bar chart comparing the proportions per sample within the total CD8+ T cell compartments of the clusters identified by Seurat or retrieved by manual gating.
N=5 per group and data are presented as mean ± s.e.m.