Subsections


Output of the Differential Expression tools


Statistical comparison tracks

The Differential Expression for RNA-Seq tool will output one or more statistical comparison tracks or tables. The statistical comparison table offers the same functionality than the track, except for the track view.

An example of a statistical comparison track is shown in figure 31.80. Statistical comparison tracks make it possible to show differential expression data alongside other kinds of tracks in a genomic context.

Image statistical_comparison_track
Figure 31.83: Statistical comparison track view.

In particular, the Fold Change value will tell you how expression levels in group 2 are relative to that in group 1.

Note that it is not possible to derive these fold changes from the CPM values by simple algebraic calculations as the Differential Expression for RNA-Seq tool works by fitting a statistical model (which accounts for differences in sequencing-depth) to raw counts.

The track layout of the statistical comparison track can be customized as follows:

The expression track table view has the following options:


Volcano plots

Statistical comparisons also offer a volcano plot view.

An example of a volcano plot is shown in figure 31.81.

Image volcano_arna
Figure 31.84: Volcano plot.

The volcano plot shows the relationship between the p-values of a statistical test and the fold changes among the samples. The log2 fold changes are plotted on the x-axis, and the -log10 p-values are plotted on the y-axis. Features of interest are typically those in the upper left and right hand corners of the volcano plot, as these have large fold changes (lie far from $ x=0$) and are statistically significant (have large y-values).

Sometimes, the volcano plot will show unexpected pattern looking like "wings", such as the ones highlighted with red arrows in figure 31.82.

Image volcanowings
Figure 31.85: Volcano plot displaying unexpected "wing" patterns.

These patterns reflect the mathematical relationship between fold change and p-value, which often becomes exposed when there are few replicates and when expression is low in one condition. For example, expression counts for two genes might be (5,5) vs (0,0) and (5,6) vs (0,1). These two genes would appear in the same "wing". Two other genes with expression counts (5,5) vs (0,1) and (5,6) vs (0,1) might be in another "wing".

When working with several samples, it can be useful to make an Expression Browser with all the samples and to open this alongside the Volcano plot. Click a point in the Volcano plot to select it and then right-click to Select Genes in Other Views. This will select the appropriate row in the expression browser.

Volcano plot side panel It is possible to change the type of p-value from the side panel (see below).

The view settings can be adjusted using the Side Panel. Under Graph preferences, you can adjust the general properties of the volcano plot

Below the general preferences, you find the Dot properties and Text format, where you can adjust the coloring and appearance of the dots and text.

At the bottom are options for choosing which values to display:

Note that if you wish to use the same settings next time you open a volcano plot, you need to save the settings of the Side Panel.