Hi,

I have a question on how the pre-ranked list of gene values should be supplied. For example, I take my results of Differential Expression between treatment and control and create a new rank statistic that takes the -log10 of the FDR value and multiplies it by the sign of the log fold change. Then I order the vector from decreasing to increasing values so the downregulated genes are at the top while the upregulated genes are at the bottom. This is shown in the code below:

newRank_pvalueAndFC = -log10(myDEresults$FDR) * sign(myDEresults$logFC)
names(newRank_pvalueAndFC) = rownames(myDEresults)
newRank_pvalueAndFC = newRank_pvalueAndFC[order(newRank_pvalueAndFC)]

In other words, the downregulated genes with a really small fdr value are at the top and the upregulated genes with a really small fdr value are at the bottom. An example of my new rank vector looks like this

Gene1   -5.2
Gene2   -3.1
Gene3   -0.1
Gene4    1.2
Gene5    3.2

The question I have is, does the ordering of the pre ranked list matter. Meaning, the way NES is calculated, do the upregulated genes have to be at the top and downregulated at the bottom so that way if the NES is positive for a pathway it corresponds to the pathway having genes active or upregulated in treatment vs control? Or can the downregulated genes be at the top as I have them and gsea will take the sign of the genes into account and even though they are found at the top, the NES score will be negative for that pathway meaning the pathway has genes enriched that are down in treatment vs control? Also, does fgsea re rank the pre ranked vector I supply it? If it does, what is the benefit of supplying it a pre ranked vector?

I appreciate any help in this as am new to GSEA and using it for enrichment.

The genes are automatically ordered within fgsea, there is no need to sort them manually.