Dear All,

i would like to ask about  a problem with two illumina  data sets i have downloaded for further analysis :

http://www.ebi.ac.uk/arrayexpress/experiments/E-GEOD-32091/?query=GSE32091

http://www.ebi.ac.uk/arrayexpress/experiments/E-GEOD-25772/?query=GSE25772

I have downloaded for each the files containing the raw data, but the txt files containing the "summary control probe profile files" are missing. Anyway, i tried firstly for the one dataset with the following functions with the R package limma:

raw.data <- read.ilmn(files="GSE32091_non-normalized.txt", other.columns="Detection")

range(raw.data$E)
[1]    20.30149 22249.08000

norm.data <- neqc(raw.data)

But after the normalization function the following appeared:

Inferred negative control probe intensities were used in background correction.

Thus, i would like to ask the two following questions:

1) The above sentence appeared due to the missing file with the control summary profiles? And if so, it is still valid to proceed with the normalization procedure and downstream analysis ??

2) Also, the minimum of my function range of my raw data, has a very small value. Thus, possibly an already background correction could have been performed ??

Yes, the message appears because you are not providing the control probe intensities. The message is to tell you that the software is figuring out what the control probe intensities were from the detection p-values. (You could have found this out by reading the neqc help page, just type ?neqc.) We thought it best to alert users when this happens, just so they are aware of what is going on.

Yes, it is still a valid way to proceed. It is virtually as good as having the control probes. We wouldn't provide the facility if we didn't think it was a valid way to proceed. And indeed, there is no other way to proceed if you want to use the neqc method and you don't supply control probes intensities.

A minimum value of 20 is not a particularly small value for a raw intensity, so I don't follow your concern about that. If Illumina global background correction had been performed, then the minimum value would have been negative.