Heatmaps

Phenotypic families

As the second analysis step, hierarchical clustering of granulocytes and monocytes-DC clusters and markers was performed and is represented as heatmaps. Heatmaps allow the visualization of the phenotype of each SPADE cluster at a glance. They represent the phenotypic diversity within the dataset, but not a particular sample and, in no case, a particular timepoint, nor do they give an idea of the abundance of each subphenotype. They are not snapshots. Clusters sharing similar phenotypes as measured by a close proximity on the cluster dendrogram, were gathered into phenotypic families. Phenotypic families were further grouped into superfamilies. This analytical strategy including 2 successive clusterings (SPADE followed by hierarchical clustering of SPADE clusters) prevents inaccurate interpretations due to potential SPADE ‘over-clustering’. Indeed, clusters may actually account for different stages of activation or maturation within a cell subpopulation, whereas phenotypic families may represent actual subpopulations.

Kinetic families

As the third analysis step, phenotypic families sharing similar dynamics were gathered into kinetic families after hierachical clustering.

Heatmap interactivity

The web favors interactivity. On mouseover and for each cluster on the heatmap, three complementary images pop-up. A cluster ID card summarizes the cluster phenotype, pedigree and dynamic. It facilitates the comparison of our data with the major well-known myeloid subsets reported to be affected by immunization. A phenotypic viewer shows the range of expression of each marker within the dataset, which is helpful to better understand and interpret the five categories color coded in the heatmap. It also shows the intensity of expression of all samples for all markers for a given cluster to assess the inter-individual variability as well as the clustering quality. Above all, a kinetic viewer shows the abundance profile over time of the kinetic family to which the cluster of interest belongs, linking phenotype to dynamic.