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Question: What doest this p.value distribution mean?
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gravatar for g.e.novakovsky
7 days ago by
g.e.novakovsky0 wrote:

Hi everyone,

I am doing an RNA-seq analysis with DESeq2. I have 3 patients with disease (affected) and 3 healthy patients (unaffected). After following standard DESeq2 pipeline (as described here), I got my table with results and p.value column. I decided to check the distribution of p.values and here it is:

I do know that we expect to see something similar to uniform distribution, apparently we don't see it here... Does it mean that there is a problem with the data or an experiment design (I used simple formula, which was taking into account genders of the patients: ~ Gender + Condition)?

Thank you

ADD COMMENTlink modified 7 days ago by Michael Love18k • written 7 days ago by g.e.novakovsky0
1

Can you show a PCA plot? This sometimes comes from batch effects, or other related correlations or differences in the variance than from the model (e.g. if one group had much larger dispersion, or an outlier).

ADD REPLYlink written 7 days ago by Michael Love18k

PCA plot is not great either... Zeros are unaffected patients, Ones are affected. PC2 divides them by gender.

ADD REPLYlink written 7 days ago by g.e.novakovsky0
3
gravatar for Michael Love
7 days ago by
Michael Love18k
United States
Michael Love18k wrote:

I guess the non-uniformity could have to do with the partial confounding of sex and condition. This seems like a good case where you could try to alleviate the distribution using fdrtool using Bernd Klaus' tutorial:

http://www-huber.embl.de/users/klaus/Teaching/DESeq2Predoc2014.html#inspection-and-correction-of-pvalues

ADD COMMENTlink written 7 days ago by Michael Love18k

Thank you, I will check it

ADD REPLYlink written 7 days ago by g.e.novakovsky0
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