TMM Normalization
The following tools automatically perform library size normalization using the TMM (trimmed mean of M values) method of [Robinson and Oshlack, 2010]:
- PCA for RNA-Seq
- Create Heat Map for RNA-Seq
- Create Expression Browser
Additionally, TMM normalization is an option in the following tools:
- Differential Expression for RNA-Seq
- Differential Expression in Two Groups
For TMM normalization, a TMM factor is computed by comparing the samples against a reference sample. The reference is the sample that has the count-per-million upper quartile closest to the mean upper quartile.
TMM normalization adjusts library sizes based on the assumption that most genes are not differentially expressed. Therefore, it is important not to make subsets of the count data before doing statistical analysis or visualization, as this can lead to differences being normalized away.