[R] limmaGUI

[J.delasHeras at ed.ac.uk]

Quoting solie.alizadeh at utoronto.ca:

> Dear all,
>
> I have a question about limmaGUI that is usually run in R environment.
> My problem is loading data into the programm. I have 6 gpr files that
> apparently are not compatible with limma. Everytime I'm trying to load
> the data (including a RNA targets file, an error appears:Error reading
> files. that I'm not sure,but seems to have something to do with the
> format of my files (gpr).
> Is that the problem? does anyone have any idea what it could be?
> I was wondering if I try GAL files, the problem would be solved. I
> still don't have access to the gal files that's why I haven't tried it
> yet.
>
> Thanks,
> Solmaz


Hi Solmaz,

for questions about BioConductor packages (such as limma or limmaGUI)  
you´re probably better off asking in the BioConductor forum. I  
recommend you subscribe to it, it´s very much microarray-oriented and  
I´ve learnt a lot there.

Now, about your question... I haven´t used limmaGUI for a long time (I  
switched to teh command line limma instead, more flexible), but I´ve  
dealt with similar problems as you describe, both my own and others...  
I can´t tell you what you´re problem is, but I´ll give you a couple of  
ideas so hopefully you can check and maybe one of them gives you a  
solution.

Well, first of all, I´m concerned that you say you have no GAL file.  
The GAL file describes what´s on the array, the locations of teh spots  
etc... and limmaGUI *requires* a GAL file, as far as I remember... so  
you won´t get far without one!
However, you say you gave GenePix files (GPR), so you could make your  
own, as each GPR file will contain the minimal info that you need in a  
GAL file: the columns with headers "block", "row", "column", "ID" and  
"Name".

The error reading the GPR files, was usually (when I encounter it) due  
to non-standard headers in teh files. If you select "GenePix" as your  
type of data file to load, by default it expects to find a column with  
the header "F532 Median" and "F635 Median" as teh source of raw  
intensity readings for the Cy3 and teh Cy5 channels respectively. But  
depending on the settings of Genepix when teh scanning was made, and  
when the quantitation was calculated, you may have a different  
wavelength (maybe 685 instead of 635, etc)... so limmaGUI cannot find  
teh right columns and returns an error. You should open all your GPR  
files (in Excel, for instance... GPR is just a standard text file, in  
tab-delimited format) and check what teh headers say. Take note of them.
If teh headers are not the same in all files, you´ll get errors.
To read GPR files with a different header than teh default, you can  
simply use teh generic option ("Other", from teh menu of file  
formats). This opens a little window where you specify teh actual  
clñumn names to load from teh files. "Rf" means "Red Foreground"  
(usually Cy5 channel), "Rb" is "Red backgrund", and similarly you get  
to specify teh column names for "Gf" and "Gb". So, say you could write  
"F650 Median" in the "Rf" slot, and this tells limmaGUI to take that  
column you specified as teh source for the signals for teh Cy5  
channel. "B650 Median" on the slot "Rb" then specifies that column  
from the GPR files as teh background for Cy5 channel... etc.

I´d say the reason above is the most likely to solve the problem.  
However, I have also seen instances where some GPR files had been  
modified to omit any empty spots, for example. This results in having  
files containing different numbers of genes, and this returns errors.  
So make sure all teh files have teh right number of rows, and that the  
GAL file has teh same number of rows too, and in the same order!

I hope this helps a bit... and do check the BioConductor forum, it is  
a great place to ask this sort of questions, especially as you are  
more likely to get answers directly from teh guys who developed the  
packages themselves.

Good luck!

Jose

-- 
Dr. Jose I. de las Heras                      Email: J.delasHeras at ed.ac.uk
The Wellcome Trust Centre for Cell Biology    Phone: +44 (0)131 6513374
Institute for Cell & Molecular Biology        Fax:   +44 (0)131 6507360
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University of Edinburgh
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