Hi, I'm relatively new to bioinformatics, and have been following the Galaxy pipleine for my RNA seq data. At this stage, I have featureCounts count tables.

In one one of the DEG experiments, I have to compare WT baseline data (2 males + 2 females) vs WT at 1h (2 males + 2 females). I am using "Timepoint" as my primary Factor, and the 2 groups mentioned before as my Factor Level 1 and 2 respectively. Thus, my primary comparison will be between these groups. However, to control for the variance in sex, from browsing Galaxy tutorials and info pages, I was drawn to the conclusion that I can introduce "Sex" as a secondary factor, and contrast 'Males' - Factor Level 1 with 'Females' - Factor Level 2. This seems a bit counter-intuitive to me, so I would really appreciate some advice on how to introduce sex as a variable in Galaxy DESeq2.

These are some examples I saw on Galaxy that pertain to this issue:

-> under "Inputs" in the DESeq2 tool on Galaxy

"The following table gives some examples of factors and their levels:

Factor Factor level 1 Factor level 2

Treatment Treated Untreated

Condition Knockdown Wildtype

TimePoint Day4 Day1

SeqType SingleEnd PairedEnd

Gender Female Male

Note: Output log2 fold changes are based on primary factor level 1 vs. factor level2. Here the order of factor levels is important."

&

https://training.galaxyproject.org/training-material/topics/transcriptomics/tutorials/ref-based/tutorial.html#analysis-of-the-differential-gene-expression

-> under "Hands-on: Determine differentially expressed features"

Thanks in advance!

István

I can't provide Galaxy or statistical analysis support, just due to time constraints, I have to focus on software-related posts. Maybe you could approach the creator of the training material.