8.3. How can I change the order of groups being compared in the Differential Expression for RNA-seq tool?
Option: All group pairs or Against control group
When running the Differential Expression for RNA-Seq tool with the option All group pairs or Against control group, the order of the comparison is study vs. control.
Hence, the Fold Change value in a statistical comparison track will tell you how expression levels in the study (the group listed before vs.) is relative to that in the control (the group listed after vs.).
- If expression values in study are twice as large as in control, the fold change will be +2.
- If expression values in control are twice as large as in study, the fold change will be -2.
The easiest way to secure that the desired group is used as control, is to choose the comparison option Against control group.
If using the option All group pairs the order of the comparison depends on the order of the input tracks selected in the tool wizard.
Thus, if selecting samples in the order 1 to 4:
You will get comparison against the lower numbers:
Whereas, if you select the samples in the wizard in reverse order from 4 to 1:
You get the comparison against the higher numbers:
Option: Across groups (ANOVA-like)
For the comparison option Across groups (ANOVA-like), the output is a single Statistical Comparison track called "Due to..."
For this comparison option, the Fold change reports the maximum pairwise fold change between any group pairs.
The order of the comparison depends on the order of the input tracks selected in the tool wizard.
For CLC Genomics Workbench version 10.00 to 12.0.x and the Advanced RNA-Seq plugin (installed to CLC Genomics Workbench 9.xx) the order of comparison for Across groups (ANOVA-like) was reverse to the order of the option All group pairs. Hence, if selecting the same samples in the same order in the wizard for the All group pairs and Across groups (ANOVA-like) options the fold changes reported by these two tests had opposite signs.
For CLC Genomics Workbench version 20 this was changed. The All group pairs and Across groups (ANOVA-like) comparisons in Differential Expression for RNA-Seq now compare expressions in the same direction. The direction is as explained for All group pairs.
Use metadata to select sample in desired order
The metadata can be used to control the order of sample selection in the Differential Expression for RNA-Seq tool wizard.
This can be done using the "Find in Navigation Area" option to have the data pre-selected when starting the wizard or by using the Right-click option to start the tool from the metadata.
Find in Navigation Area option:
Launch tool from metadata table option:
In the example below the following metadata was used to control the order of the samples for analysis with the All group pairs option: