Single Cell UMAP/tSNE Viewer

The UMAP/tSNE viewer displays the cells in a dimensional reduction space (UMAP or tSNE). These coordinates are obtained during Clustering or Trajectory analysis. The cells (dots) can be colored by different features. Moreover, they can be selected and added to new custom groups (Figure 1).

Figure 1. Interactive UMAP/tSNE viewer.

Adjustments

This section modifies the general appearance of the plot, that is, the coordinates and the color of the cells.

Projection. Select which dimensional reduction space to plot: UMAP or tSNE (Figure 2). This will change the coordinates of the cells.
Color by. The type of feature to color cells by (Figure 3). Depending on the category chosen, the configuration below will change.
Cell Metadata. Color cells by the output of the Clustering or Trajectory analyses. These cell metadata groups can’t be modified.
My Classifications. Color cells by the output of Cell Type Annotation or custom groups. These cell metadata groups can be modified.
Gene Expression. Color cells by the expression of one or multiple genes.
Pseudotime. Color cells by pseudotime values. This is only available after Trajectory analysis.

Figure 2. Select plotting the coordinates of the UMAP or tSNE in the "Projection".

Figure 3. Different options of the "Color by" parameter.

Cell Metadata

This category includes groups obtained by Clustering or Trajectory results (Figure 4-A). Clustering results include the "seurat_clusters" groups. Trajectory results include "monocle_clusters", "monocle_partitions", and "ptime_ranges" groups. The levels appearing on the Side Panel and the plot’s legend will change according to the selected group (Figure 5).

The color of the cells belonging to those groups can be modified by clicking on the corresponding color square and selecting a new color (Figure 4-B).

Figure 4. Cell Metadata groups (A) and how to modify the color of a group (B).

Figure 5. UMAP with cells (dots) colored by the "seurat_clusters" group in the Cell Metadata category.

My Classifications

This category includes groups generated after Cell Type Prediction or by the "Assign Group to Selection" tool (explained below) (Figure 6). The characteristic of this category is that both groups and levels can be renamed or deleted (Figure 7).

Cells will be colored attending to the label assigned in the selected Group. The color of a given label can be modified by clicking on the corresponding color square and selecting a new color (Figure 4-B).

Figure 6. UMAP with cells (dots) colored by results of the Cell Type Prediction tool.

Figure 7. Modification buttons for the group and level names for the My Classifications category.

Gene Expression

This category will change the display of the section below. Cells will be colored according to the expression of the specified gene(s) (Figure 8).

Genes to plot can be specified on the "Gene" box (Figure 9-A). Gene name suggestions will appear while typing. Alternatively, a gene list can be pasted on the box.

More than one gene can be specified while plotting. In this case, it is possible to select how to summarize the expression values for each cell: by computing the average, the sum of all expressions, taking the minimum value, or taking the maximum value (Figure 9-B).

Figure 8. tSNE visualization with cells colored by the average expression of the specified genes.

Figure 9. Color cells by Gene Expression category. A - Feature name suggestions appear while typing. B - Statistics to group gene expression levels for an individual cell.

Pseudotime

This category will change the display of the section below. Cells will be colored according to their pseudotime value (Figure 10). This option will be available only after Trajectory Analysis.

The color of each state (Initial, Intermediate I, Intermediate II, and Final) can be modified by clicking on the corresponding color square and selecting a new color.

Selection

This section allows selecting cells directly on the UMAP/tSNE. The selection tools are available independently of which category is selected in the "Adjustments" section.

Once the selection is done, the cells can be assigned to a new group. There are different options for performing the selection:

Lasso. It allows drawing free-form shapes to select and highlight specific points or areas of interest (Figure 11).

Figure 11. Example of the "lasso" tool for selecting areas of the UMAP/tSNE viewer.

Rectangular. Draw a rectangular shape to select an area of interest (Figure 12).

Figure 12. Example of the "rectangular" tool for selecting areas of the UMAP/tSNE viewer.

Horizontal. Select cells based on the horizontal axis, that is, by the values on the X-axis (Figure 13).

Figure 13. Example of the "horizontal" tool for selecting areas of the UMAP/tSNE viewer.

Vertical. Select cells based on the horizontal axis, that is, by the values on the X-axis (Figure 14).

Figure 14. Example of the "vertical" tool for selecting areas of the UMAP/tSNE viewer.

Multiple selection. Clicking this button allows selecting multiple areas of the plot. If this option is not activated, a new selection will overwrite the previous one (Figure 15).

Figure 15. Example behavior of the "multiple" tool in the UMAP/tSNE viewer.

Clear. This button removes the actual selection.
Invert. This button reverses the selection process, deselecting the points or areas previously selected and selecting all other points or areas that were not initially included (Figure 16).

Figure 16. Example behavior of the "invert" tool in the UMAP/tSNE viewer.

Remaining. If a group with unclassified cells is displayed in the "Adjustment" section, clicking this button selects all cells that have not been classified (Figure 17).

Figure 17. Example behavior of the "remaining" tool in the UMAP/tSNE viewer.

Advanced Selection

This section allows specifying rules for selecting the cells, instead of selecting them directly on the plot. It is possible to apply both categorical (based on group identity) and/or based on gene expression levels (Figure 18).

One or several categorical and/or expression rules can be applied at the same time. In this case, the "Join rules by" parameter controls if the rules will be joined with AND or OR.

Click on the buttons "Add" next to the "Categorical Rules" and "Expression Rules" parameters to specify a new rule.

For adding a categorical rule, select if the cells must be IN or NOT IN the group and label selected in the following dropdown lists.

For adding an expression rule, type a gene name, select a criterion (greater than >, equal to =, not equal to !=, etc.), and type the threshold value.

After all the rules have been specified, click on Apply to make the selection. If a new rule is added or removed, the Apply button must be clicked again.

Figure 18. Example behavior of the "Advance Selection" section. Cells belonging to the "Root endodermis" group and with an AT3G55230 expression level greater than 1 are selected.

Assign Group to Selection

Once cells have been selected with the tools explained above, they can be assigned to custom groups in the "Assign Group to Selection" section (Figure 19).

The selection can be added to a preexisting group/value or a new group and/or value can be created by typing on the corresponding box.

The new group and value will be available in the "My Classifications" category in the "Adjustments" section.

Figure 19. Example of how to add selected cells to custom groups.

Font & Graphical Settings

This section controls the general settings of the plot appearance. E.g: font family and size for each axis and title, the size of the dots, wether to include grids or not, etc.

Figure 20. Font and graphical general settings.