correct order for pathway analysis

Question

[Sara]

I am doing pathway analysis but there a lot of tutorials so, I am looking for the correct way of doing that.

1- I got DE genes (up and down regulated)
2- filter them based on log2 of FC. only the genes with Log2FC > 1 or Log2FC < -1 are used for pathway analysis
3- I used up and down regulated genes together (only gene name) for pathway analysis (using DAVID) to identify up and down regulated pathways.

the question is that, ma I following the correct order? if not what is the correct order and tool?

[JC]

What is your reasoning for combining up/down in 3?

The list of down- and up-regulated genes indicates which genes are being affected by your treatment, the combination of both are representing all pathways that could be affected, but you cannot separate up- from down-regulated pathways.

In my opinion, it's better to perform pathway enrichment separately, with the main hypothesis: if several or key genes are up-regulated and are part of a pathway, the pathway is also up-regulated, similar hypothesis for down-regulated.

[dsull]

I agree with what JC said. Additionally, I recommend using an updated, well-maintained pathway tool such as enrichr: https://maayanlab.cloud/Enrichr/ instead of DAVID.