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Differential expression analysis can be used to compare cell types. Here, we will compare glioma and oligodendrocyte cells to identify genes differentially regulated in glioma cells from the oligodendroglioma subtype. Glioma cells in oligodendroglioma are thought to originate from oligodendrocytes, thus directly comparing the two cell types will identify genes that distinguish them.
Filter cells
To analyze only the oligodendroglioma subtype, we can filter the samples.
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The filter lets us include or exclude samples based on sample ID and attribute.
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A Filtered counts data node will be created with only cells that are from oligodendroglioma samples (Figure 3).
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Identify differentially expressed genes
- Click the new Filtered counts data node
- Click Statistics > Differential analysis in the task menu
- Click GSA
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Next, we will set up a comparison between glioma and oligodendrocyte cells.
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- Click Finish to run the GSA
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Because of the large number of cells and large differences between cell types, the p-values and FDR step up values are very low for highly significant genes. We can use the volcano plot to preview the effect of applying different significance thresholds.
- Click
to view the Volcano plot - Open the Style icon on the left, change Size point size to 6
- Open the Axes icon on the left and change the Y-axis to FDR step up (Glioma vs Oligodendrocytes)
- Open the Statistics icon and change the Significance of X threshold to -10 and 10 and the Y threshold to 0.001
- Open the Select & Filter icon, set the Fold change thresholds to -10 and 10
- In Select & Filter, click
to remove the P-value (Glioma vs Oligodendrocytes) selection rule. From the drop-down list, add FDR step up (Glioma vs Oligodendrocytes) as a selection rule and set the maximum to 0.001
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We can now recreate these conditions in the GSA report filter.
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The filter should include 291 genes.
- Click
to apply the filter and generate a Filtered Feature list node
Exploring differentially expressed genes
To visualize the results, we can generate a hierarchical clustering heatmap.
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- Double click the green Hierarchical clustering node to open the heatmap
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- Click Glioma (multi-sample) to return to the Analyses tab.
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A Gene set enrichment node will be added to the pipeline .
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| Additional assistance |
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