Hello,
I am using limma to determine differential gene expression between healthy and KO mice.
In my design matrix, I am including several covariates that I know influence gene expression, but that I am not interested in.
Specifically, it looks something like this: 0+disease_status+age+batch+onset.
For disease_status there are only two value (diseased or healthy) and onset describes the site where the first symptoms occurred.
The problem is, that for all healthy animals, the value for onset is "Undefined", because obviously there is no site of onset because they are healthy.
This basically means, that the "healthy" samples of disease_status are confounded with onset. I think that is the reason why I get the following warning when I run limma:
Coefficients not estimable: ...
Is there a way to adjust the design matrix so this problem does not occur or how should I handle this issue?
Any insights are much appreciated!
