DESeq2 for multiple groups

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Hi everyone,

I am performing differential expression analysis using featureCounts files that I previously analyzed in UseGalaxy. However, I am now very confused. Does DESeq2 in UseGalaxy allow for comparisons such as (Overexpression cell line1 - Control1) - (Overexpression cell line2 - Control2)? I have two different cell lines, each with its own control, and I want to compare them. From what I understand, DESeq2 uses the first factor as the reference. What if I have two reference groups? In this case, would Limma-voom be a better option?

Another question: If I have two or three treatments but only one reference (control) and want to compare the treatments with each other, how should I set up the analysis? All treatments come from the same cell line, but they are part of different experiments, all sharing the same control. For example, I have cell line 1 with treatment 1, treatment 2, treatment 3, and a common control. How can I properly compare the treatments with each other?

Thanks in advance, Bana

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Welcome @Bana

With DESeq2, yes, you can combine different levels and the first factor will be the reference, and use of Limma will allow for the use of additional batch organization with a “contrast”.

Links to tutorials with simple example usage is on each tool form with the Bioconductor vignettes as the definitive resource. Questions can also be asked at their support forum, example. Remember that you can enter an R environment in Galaxy to use these tools in more complex ways than the tool form may support.

Beyond that, please consider consulting a statistician to ensure the correct experimental design and result interpretation, especially if this is for a publication!

Hope this helps! :slight_smile:

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Thank you Jennaj for you reply. Indeed it is very helpful!

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