Using limma to read Illumina HT-12 v4 data
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@99c42105
Last seen 5 weeks ago
United Kingdom

hiya, I'm an undergrad who's completely new to bioinformatics and R. I've been assigned a project to analyse the differential gene expression from some Illumina HT-12 v4 data on GenomeStudio but when I try to follow the limma user guide, I run into errors right at the start when I try to use neqc:

x <- read.ilmn(files="Diff Expression Probes.txt", ctrlfiles="Control Probe Profile.txt")

y <- neqc(x)

Note: inferring mean and variance of negative control probe intensities from the detection p-values.
Error in if (alpha <= 0) stop("alpha must be positive") : 
  missing value where TRUE/FALSE needed
In addition: Warning message:
In min(d) : no non-missing arguments to min; returning InfY<-neqc

I thought maybe I had exported the wrong data from GenomeStudio since the limma user guide says "probe profile.txt" but I only found "Diff. Expression Probes.txt" on GenomeStudio? Or is there some deeper error I haven't noticed? I don't know what it means by alpha either?

Thanks for your help!

limma • 139 views
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I edited the title of your question to be more specific.

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@gordon-smyth
Last seen 3 hours ago
WEHI, Melbourne, Australia

It seems clear that you haven't exported the correct intensity data from GenomeStudio, and I think you recognize that yourself in your question. I don't know what the file "Diff. Expression Probes.txt" contains, but it seems from the name of the file that it is output from a differential expression analysis. That obviously can't be right. read.ilmn is intended to read raw probe intensities. If you don't have the raw intensities from GenomeStudio, then you can't use read.ilmn and neqc.

To troubleshoot you need to open the files in an editor to see what they contain. If you want to see what the intensity files should look like, the files analysed in the limma User's Guide are available here: https://bioinf.wehi.edu.au/marray/IlluminaCaseStudy/

The function read.ilmn was written to handle Illumina beadchip files 15 years ago. Nowadays we have the ability to read the Illumina IDAT files instead. These days, it would be better to read the IDAT files using read.idat, and then feed the read.idat input into neqc. Type help("read.idat") at the R prompt.

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