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. 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".

Image experimental_design
Figure 26.25: Setting up the experimental design and comparisons.

This will display the wizard shown in figure 26.25.

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

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).

How many replicates do I need? The Differential Expression for RNA-Seq tool is capable of running without replicates, but this is not recommended and the results should be treated with caution. In general it is desirable to have as many biological replicates as possible - typically at least $ 3$. Replication is important in that it allows the `within group' variation to be accurately estimated for a gene. In the absence of replication, the Differential Expression for RNA-Seq tool assumes that genes with similar average expression levels have similar variability.

Technical or biological replicates? [Auer and Doerge, 2010] illustrates the importance of biological replicates with the example of an alien visiting Earth. The alien wishes to know if men are taller than women. It abducts one man and one woman, and measures their heights several times i.e. performs several technical replicates. However, in the absence of biological replicates, the alien would erroneously conclude that women are taller than men if this was the case in the two abducted individuals.