**20**wrote:

I'm analyzing an RNAseq with the following samples/groups:

`A = 14 samples, B = 7, C = 7, D = 10 and E = 10.`

Each sample is independent from another and represents a biological sample from one patient with a different form of a disease X.

I've analysed this experiment with DESEq2 with the "standard" workflow provided in the vignette.

Following those instructions, based in the DESeq() function, I've found a few to **none** DEG from pairwise contrasts, such: A vs B, C vs A etc, with an adjusted P-value <= 0.05 (FDR for multiple testing correction).

As suggested by Klaus (http://www-huber.embl.de/users/klaus/Teaching/DESeq2-Analysis.pdf), I checked the p-values distribution with an histogram and it deviates from an expected distribution (uniform dist. with a peak at 0, under the null). Then, I employed a correction using the package fdrtool using the following code:

`res.de.fixed <- fdrtoolres.de$stat, statistic = "normal")`

Atfer that, I've gotten an expected p-value distribution and replaced my old adjusted p-values with the new **qvalues** obtained with fdrtool. Now, as expected, I have a lot of DEG with alpha = 0.01, and they seem to make sense given previous experiments with microarray.

My question is: **Is this a legit approach?** Can I correct the p-values obtained from Negative Binomial GLM fitting and Wald statistics employed by the DESeq() function with fdrtool? Of course, when the p-values exhibit this strange distribution. **Also, what's the difference between the q-values (fdr) and the local fdr (lfdr) from fdrtool?**

**The histograms:**

Top, before correction with fdrtool. Bottom, after fdrtool code above.

**Edit.2. **Added PCA from DESeq2::plotPCA()

From this PCA, I can see some underlying hidden effect, maybe a batch or lane effect? I'll need to get these data from the source.

Thanks.

Thyago

dear Thyago,

A few other things to check: can you supply a PCA plot of the samples, and additionally, can you confirm that you did not use an LFC threshold when testing with results().

14kDear Michael,

Added. I confirm not using any lfc cutoff from results() function. I used the default, 0, lfc.

Thank you.

20