Hi! I am conducting an RNA-seq experiment. After performing a functional enrichment analysis to identify significant biological processes, I have identified contradictory gene-ontology (GO) terms related to cell cycle in the same group. The yellow group suggest positive regulation of mitosis, while the pink group suggest negative regulation of mitosis.
How would you interpret these contradictory data?
Thanks in advance!
This is normally observed in whole transcriptome RNA-seq data analysis, and there are multiple reasons for this, including the one very basic factor that how pure the tissue in question was prior to the RNA isolation. The RNA-seq data (FPKM) usually falls in the negative binomial distribution in which the variance is higher than the means among the biological replicates. So, applying suitable filtering strategies and data normalization can minimize or eliminate such outcomes in the data.
At the data analysis level, multiple tools can be used for differential expression of genes, instead of relying on a single tool (this is because some DEG tools assume the read counts data as parametric, whereas, others assume non-parametric). Also, the genes set enrichment tools such as DAVID also reports enrichment of gene with P or FDR > 0.05. Such enrichments should not be considered. Ideally, the genes with FPKM values >= 0.5 are considered for the downstream analyses.
Furthermore, some genes have dual or multiple types of biological roles depending upon the types of tissue. You can manually check the roles of contradictory genes using the GeneCards database.
Good Luck
Your results are not contradictory. Red processes are only more specific (down in the ontological tree) than those on the left, which you are wrongly interpreting as "positive" processes while they are only less detailed.
GO longitudinally describes biological processes according to their degree of generality. You can check this using Amigo2 or similar tools and searching one of your terms, e.g., "negative regulation of cell cycle process" in the Inferred Tree View. An example is attached.
You can safely consider your processes together.
Thank you Tommaso Mazza for your answer. I see what you mean. I checked the relationship between these GO terms with Amigo2 (see attached). I can distinguish two branches in the tree, but I don't strictly see the yellow processes to be lower in the tree compared to the pink ones. Are they not in parallel levels? Would you rather say that pink are more specific because they indicate a specific direction for the regulation (negative)? In that case, I must interpret as negative regulation of the cell cycle, isn't it?
Thank you!
Thank you Muhammad Shakeel for the detailed response and information.
Terms whose names are prepended with "negative" or "positive" words are lower in the GO terms than those without the specification of the regulation direction. I was saying that your results are more detailed for some terms than others. This happens frequently, and it is not a sign of any incongruence in your results.
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