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.