Hello.
I'm currently in the process of conducting a gene ontology enrichment analysis and I'd greatly appreciate your insights on a strategic decision. The question at hand revolves around whether to consider all genes with a padj < 0.05 for the analysis, or to adopt a more refined approach by segregating the analysis into two distinct groups based on log2FC thresholds and the padj value.
Here are the two approaches I'm considering:
Approach 1: Inclusive Analysis I could include all significant genes, those with a padj < 0.05, regardless of their log2FC values. This approach provides a comprehensive view of the entire range of biological changes occurring in the dataset.
Approach 2: Separate Analyses for Upregulated and Downregulated Genes Alternatively, I could perform separate analyses for genes meeting different log2FC thresholds. Specifically, genes with a padj < 0.05 and log2FC > 2 would constitute the 'upregulated' group, while those with a padj < 0.05 and log2FC < -2 would form the 'downregulated' group.
I'm particularly interested in your thoughts on these options, considering your expertise. Could you please provide insights on which approach might be more suitable for gaining meaningful insights into GO-enrichments and pathways analysis?
Please feel free to share your thoughts, and I'm open to any additional suggestions you may have. Thank you in advance for your guidance.
You could do both with the understanding you are asking different questions. There are many gene sets that are defined as 'down-regulated' or 'up-regulated', so your 2nd approach may make more sense with these, your first approach may make more sense when looking at more general gene sets.
A log2FC of 2 or -2 seems like a pretty high threshold, usually you could do 0.58, 1, or just positive/negative thresholds.
Either way, I wouldn't spend too much time on this type of analysis. You can usually find something, or anything, in the gene sets and I think trying to derive too much information from this analysis can be a waste of time. Ultimately you will want to validate or, at the very least, have orthogonal / complementary results, so I think the goal is to get to that point sooner rather than later.