Differential Expression for RNA-Seq

The Differential Expression for RNA-Seq tool performs a statistical differential expression test for a set of Expression Tracks. It uses multi-factorial statistics based on a negative binomial GLM. The tool supports paired designs and can control for batch effects. The statistical analysis is described in more detail in The statistical model.

To run the Differential Expression for RNA-Seq analysis:

        Toolbox | RNA-Seq Analysis | Differential Expression for RNA-Seq

Select a number of Expression tracks (Image rnaseqtrack_16_h_p) and click Next figure 28.27.

Image expressionrnaseq
Figure 28.27: Select a number of Expression tracks.

For Expression Tracks (TE), the values used as input are "Total transcript reads". For Gene Expression Tracks (GE), the values used depend on whether an eukaryotic or prokaryotic organism is analyzed, i.e., if the option "Genome annotated with Genes and transcripts" or "Genome annotated with Genes only" is used. For Eukaryotes the values are "Total Exon Reads", whereas for Prokaryotes the values are "Total Gene Reads".

Note that the order of comparisons can be controlled by changing the order of Expression track inputs.

The normalization options available can be seen in figure 28.28.

Image normamethod
Figure 28.28: Normalization methods.

First, choose the application that was used to generate the expression tracks selected at the previous step: Whole transcriptome RNA-Seq, Targeted RNA-Seq and Small RNA. For Targeted RNA-Seq and Small RNA, you can choose between two normalization methods: TMM and Housekeeping genes, while Whole transcriptome RNA-Seq will be normalized by default using the TMM method.

TMM Normalization (Trimmed Mean of M values) calculates effective libraries sizes, which are then used as part of the per-sample normalization. TMM normalization adjusts library sizes based on the assumption that most genes are not differentially expressed.

Normalization with Housekeeping genes for can be done when a set of housekeeping genes to use is available: in the "Custom housekeeping genes" field, type the name of the genes separated by a space. Finally choose between these two options:

When working with Targeted RNA Panels, it is recommended to normalize with the Housekeeping genes method rather than TMM. Predefined list of housekeeping genes are available for samples generated using Human and Mouse QIAseq panels (hover with the mouse on the dialog to find the list of genes included in the set). If you are working with a custom panel, you can also provide the corresponding set of housekeeping genes in the "Custom housekeeping genes" as described above.

In the Experimental design panel (figure 28.29), a Metadata table must be selected that describes the factors and groups for all the samples.

Image experimental_design
Figure 28.29: Setting up the experimental design and comparisons.

The Comparisons panel determines the number and type of statistical comparison tracks output by the tool (see Output of the Differential Expression for RNA-Seq tool for more details).

The Differential Expression for RNA-Seq tool produces different numbers and types of statistical comparison tracks depending on the settings of the Comparisons panel. Depending on the choice either a Wald test or a Likelihood Ratio test is used. For example, assume that we test a factor called 'Tissue' with three groups: skin, liver, brain.

Note: Fold changes are calculated from the GLM, which corrects for differences in library size between the samples and the effects of confounding factors. It is therefore not possible to derive these fold changes from the original counts by simple algebraic calculations.