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Housekeeping gene normalization is never applied by the tool Differential Expression in Two Groups

Issue description

The issue described here affects the tool Differential Expression in Two Groups

In affected software, normalization always proceeds using Normalization method = TMM, even when Normalization method = Housekeeping genes has been selected as in the screenshot below.

Effect on results

The choice of normalization method affects the calculated fold changes and p-values. In many cases, the two normalization methods will give consistent results, such that there is a very large overlap in the number of differentially expressed genes (defined by a combination of fold change and p-value cut-off).

However, results can differ substantially when the assumptions of either normalization method are violated:


It is recommended to re-run affected data with the tool Differential Expression for RNA-Seq, which is not affected by this issue. Old and new results can be compared using Create Venn Diagram for RNA-Seq.

Affected software

This issue affects:

This issue was addressed in version 21.0.5 of the software.