I am trying to use the limma-voom tool and I have been following the “Genes to Counts” training. Within the training it gives the tip about “multiple factors” but the tip does not give an example of how the input would change when entering the “contrast of interest”. I have tried various different inputs to try and figure out on my own but when I do, I always get an error and from the errors it seems that the program will not read pass the 2nd column of the either the factor file or when inputted manually.
What I am trying to do here is take all of the samples from the “Reaction” factor that are in group “A” and contrast the “Treatment” factor of groups “T-P”. I continue to receive an error stating that object “P” cannot be found which leads me to believe that the program is only reading the 1st column (sample IDs) and the 2nd column (the “Reaction” factor. I tried entering the multiple factors and groups manually but either way on the first factor and groups are accessed.
Can someone please assist me with either how to input the “contrast of interest” to account for multiple factors and groups or can some explain it to me differently (maybe I am missing something here)?
Correct, the contrasts string can only contain one - per grouping. Why? Those are interpreted.
You could input two different lines, or two different blocks on the form. Or create a compound term with parenthesis. The form has examples of this right under each input area, plus a link to the Bioconductor manual (these terms are passed directly to the underlying tool, so the formatting is identical).
And, maybe your example was simplified … but is there a reason why all treatment groups couldn’t have the same designation for this run? Meaning, the second column of your file could have just T for all. That would simplify your input. If you are using collections with group tags, it would be pretty quick to relabel the group tags for a collection that way (See Hands-on: Group tags for complex experimental designs / Using Galaxy and Managing your Data).
I do however, have an additional question. When creating the factor file, how do you incorporate your groups in this file? It is my understanding that the 1st column is to be your samples (matching the count file), and the second column is to be your primary factor and any additional columns will count as additional factors. How do I include the names of the groups for the factors in the factor file?
Yes, thank you for this clarification. What I am attempting to do is use the limma-voom tool and I am wanting to create a excel factor file. I have gone over the “RNA-seq counts to genes” tutorial, but what it does not explain is how do I include the groups in this file.
For example, if you use the snippet of the factor file I provided earlier, you see the 1st column are the samples, the 2nd “factor 1”, and the 3rd
“factor 2”. I would like to include the groups “group1” & “group2”.
I am I following your explanation correctly as to do this the factor becomes the title header followed by the group to achieve this?
but let me know if you have any questions.