Hi, I’m trying to combine two technical replicates (featureCounts: counts file (tabular)) together for DESeq2 but didn’t find the right function to do so. (i.e., I have 6 samples in total; for each sample I have 2 technical replicates, and my previous analysis with DESeq2 treats the samples as 2 factor level (6 WT 6 KO). While now I need to combine each replicates into one sample for analysis (i.e. 3WT 3KO). Could anyone help on this? (which join or combine function should I use?) Thanks a lot.
Dear @GU111,
Technical replicates can typically either summed or averaged as mentioned in the literature [1] [2] [3] and several posts [4] [5].
I would do it quickly on the terminal. However, in Galaxy maybe is best to join the two count file for each replicate pair with [Join two files] and then apply [Compute an expression on every row].
If you calculate the average between replicates, then make sure none of them is a heavy outlier. The best practice to check for this is to calculate the sample correlation between all replicates. Each pair of technically replicates should have a high correlation. I am not completely sure about a good threshold, because it depends on your experiment. But you can be certain that you have no heavy outliers if each correlation is |p| > 0.8.
I hope I could help and best wishes,
Florian
Hi Florian, thank you for your reply. I think i need to get the mean of the technical replicates of the count. However, when I used the join function, it turns out to give me wrong results. Would you let me know what the right function that I should use to get the mean counts of the genes of two featureCounts count files?
Dear @GU111,
As fasr as I know, there is no simple “mean function” in Galaxy. That is why you first have to join your two replicate feature count files with [ Join two files] and then apply [ Compute an expression on every row] to calculate the mean of the two columns in your new result file from [ Join two files].
Cheers,
Florian