Differential Expression for Single Cell

Differential Expression for Single Cell detects differentially expressed features using expressions from an input Expression Matrix (Image expression_matrix_track_16_n_p) / (Image expr_matrix_spliced_unspliced_16_n_p) and groupings provided by Cell Clusters (Image cell_clusters_16_n_p) or Cell Annotations (Image cell_annotations_16_n_p).

It is often most natural to run the tool from a Dimensionality Reduction Plot by right-clicking on the plot, see UMAP and tSNE plot functionality for details. However, it can also be found in the Toolbox here:

        Gene Expression (Image sc_gene_expression_folder_open_16_n_p) | Expression Analysis (Image sc_expression_folder_open_16_n_p) | Differential Expression for Single Cell (Image sc_differential_expression_16_n_p)

The tool tests if each feature is differentially expressed and outputs Statistical Comparison Tables (Image sc_stat_comp_16_n_p).

The first set of options narrow down the focus of the tool:

It is easiest to understand the effects of these settings with example data from figure 7.1. If the table shown there were supplied as either `Clusters' or `Cell annotations', then the possible values of `Test differential expression due to' would be `Sample', `Status' or `Cell type' (the `Barcode' column is special and is excluded). If `Cell type' were chosen, then possible groups in `Select groups' would be `T cell', `B cell' and `Platelet'.

Image experimentsetup
Figure 7.1: Example data consisting of cells with different cell types coming from either Case or Control samples

From now on, we will continue with this example, assuming that Test differential expression due to = Cell type. There are two possible types of tests: `All group pairs' and `Identify marker genes'.

All group pairs

In the example, there are three groups: `T cell', `B cell' and `Platelet'. When All group pairs is selected, up to 6 pairwise comparisons can be performed. Only three of these will be output, for example `T cell vs B cell', `T cell vs Platelet', and `B cell vs Platelet'. The other three tests, `B cell vs T cell', `Platelet vs T cell', and `Platelet vs B cell' will not be produced - this is because the only difference between, for example, `T cell vs B cell' and `B cell vs T cell' is the sign of the fold change.

It is possible to control exactly which comparisons are performed by using the Select groups option. The order of any selected groups determines the direction of the comparisons. For example, if Select groups = Platelet, B cell, T cell, then the comparisons will be `Platelet vs B cell', `Platelet vs T cell', and `B cell vs T cell'. If Select groups = T cell, B cell, Platelet, then the comparisons will be `T cell vs B cell', `T cell vs Platelet', and `B cell vs Platelet'.

The Select groups option can also be used to restrict the number of comparisons. If Select groups = B cell, Platelet, then the outputs will be reduced to just those involving the selected groups. In this case there would only be one output: `B cell vs Platelet'.

Identify marker genes

In the CLC Single Cell Analysis Module, marker genes are considered to be genes that are differentially expressed in the group of interest when compared to all other groups. This does not necessarily mean that they are only expressed in the group of interest, or are up-regulated in the group of interest; marker genes may also have abnormally low expression (though this is unlikely), or have an expression that, by being lower than in some groups and higher than in others, is distinctive to the group of interest.

In practice, the requirement that marker genes are differentially expressed compared to all other groups can be overly strict. For example, a group might contain so few cells that it is never possible to detect differential expression compared to this group. To avoid this problem, groups are excluded if they have no significant differentially expressed genes relative to a majority of the other groups. Here, significant means that the FDR p-value is less than 0.05.

Select groups determines the groups for which the markers have to be differentially expressed. For example if Select groups = Platelet, B cell, T cell then three sets of markers will be output `Platelet vs rest', `B cell vs rest' and `T cell vs rest'. The markers for `Platelet vs rest' will only be differentially expressed when compared to B cells or T cells - if there was another cell type in the data that had been excluded from the selected groups, then it is possible that the markers in `Platelet vs rest' would not be useful for distinguishing platelets from this additional cell type.

Marker genes are identified by first running `All group pairs' and collecting the pairwise results into marker results as detailed above.

Performing separate tests between conditions for each cell type

It is possible to make comparisons between conditions (e.g. Case vs Control) for each cell type using the option Perform a separate test for each group in. Again this is easiest to illustrate with reference to figure 7.1.

Using `All group pairs' with Test differential expression due to = Status and Perform a separate test for each group in = Cell type will give the outputs `T cell: Case vs Control', `B cell: Case vs Control', and `Platelet: Case vs Control'.

Selecting genes to be tested

When a gene is expressed in too few cells, there could be too little information to reliably detect if it is differentially expressed. A minimum number of cells expressing the gene can be set using Minimum number of cells and Minimum percentage of cells. A gene is considered to have insufficient expression in a group if one of the following is true:

When a pairwise comparison is performed, tests are not performed for genes with insufficient expression in both groups, and the p-value is set to NaN (not a number).

This also affects `Identify marker genes', as the markers are obtained from pairwise tests. For markers, the test for a gene is not performed when the group of interest and at least one other group have insufficient expression.