Dear All,

I have analysed gene expression microarray results using limma and have a list of pvalues. I want to use qvalue to look for the FDR. Every time I try to use qvalue I get the error : "ERROR: The estimated pi0 <= 0. Check that you have valid p-values or use a different range of lambda."

I have repeated my limma analysis and end up with the same results. The p values look OK to me and are all within the range 0-1. My working is as below:

library(qvalue)
> setwd("~/Desktop/mRNA arrays/")
> pvalues=read.table("pvalues.txt")
> range(pvalues)
[1] 0.000299344 0.753304443
> qobj<- qvalue(p=pvalues)
Error in pi0est(p, ...) :
  ERROR: The estimated pi0 <= 0. Check that you have valid p-values or use a different range of lambda.
>

Could anyone please explain why I am getting this message?

Thanks,

Clara Green

Clinical Research Fellow

University of Birmingham, UK

The problem comes up because your range of p-values appears to be truncated at about 0.75. The expected range of p-values is (0-1], and the fact that you are missing a whole quartile of expected values is throwing the pi0est() function for a loop. The 'fix' is given in the error message, saying you need a different range of lambda (the 'stock' range is 0.05-0.95, so you get problems because your p-values have a narrower range). As an example:

> ps <- runif(3e5)
> library(qvalue)
> ps <- ps[ps < 0.75]
> qs <- qvalue(ps)
Error in pi0est(p, ...) :
  ERROR: The estimated pi0 <= 0. Check that you have valid p-values or use a different range of lambda.
> qs <- qvalue(ps, lambda = seq(0.05, 0.75, 0.05))
>

But a better question is why you have this weird distribution of p-values. That sounds pretty questionable to me, and would be worth looking into.

limma already provides you with FDR values. Just call topTable() and FDR values will be in the column adj.P.Value.

I doubt that you will need this, but limma also provides ways to estimate pi0 without getting errors. See ?propTrueNull.