Dear all,
The drug effects are so drastic that the cell lines have more than 75% of the genes differentially expressed (3 control vs 3 treatment). I used the TMM normalization and EdgeR for the analysis, which assume a maximum of 60% of differentially expressed genes. How to proceed with such a case? Should I trust the results if the assumptions are violated?
Thanks,
Aaron, the logratioTrim is a parameter of edgeR I can change ?
The option of adding spike-ins is not duable right now, would it be a proper approximation of trusting only DE genes with extreme fold change i .e FC >2 or FC>4 ?
1) You can set
logratioTrim
as an argument incalcNormFactors
.2) The problem is that you don't know how wrong your normalization is. Consider an example where most genes decrease in abundance by 10-fold in your treated cells. After normalization, the majority of genes would appear to be non-DE, and you would instead observe 10-fold "upregulation" for genes that did not change in abundance. So it's hard to say whether an extreme log-fold change is likely to be correct when the normalization cannot be trusted.