[BioC] Limma and limmaGUI with Imagene files, problems/bugs report

[STKH (Steen Krogsgaard)]

Hi,

we routinely analyze Imagene files with BioC, so perhaps I can answer
some of your problems.

First, you have to define one metagrid for all your spots, BioC won't
understand fields. We have a 4x6 grid spotted twice on the slides, so we
have defined a 4x12 metagrid. I guess you can modify your imagene output
text files in e.g. excel to cope with the grid problem, but I find that
tedious. So define your grid in Imagene once and for all.

readTargets: I do it exactly the way you describe, first readTargets and
the matrix. Is that a problem? It's just two lines of code...

Spottypes: Be sure that the heading of your ID column in the spottypes
file is "Gene ID" (exact spelling but without quotes), then
controlStatus (which I assume you meant when you wrote controlTypes)
will match to the right column in RG (RG at genes@Gene ID).

Hope this helps
Steen


-----Original Message-----
From: bioconductor-bounces at stat.math.ethz.ch
[mailto:bioconductor-bounces at stat.math.ethz.ch] On Behalf Of Dr. Ir. B.
van Breukelen
Sent: 1. december 2004 13:23
To: bioconductor at stat.math.ethz.ch
Subject: [BioC] Limma and limmaGUI with Imagene files, problems/bugs
report


Dear limma users and developers,

At our laboratory we analyze our MA data using limma. Currently we have
created a dedicated array on which the same set of genes is spotted
three times on one slide. Actually it is one design spotted three times.
In Imagene you can now create a META gid for (in this case) the 4*4
grids. This grid you call grid A.. Then you can copy it several times
(thereby creating metagrid B and C). You then end up after
quantification with a large imagene file having the information of the
three metagrids: (its like having three slides on one slide!) e.g.
Field	GridRow	GridCol	SpotRow	SpotCol	ID
A	1		1		1		1
Pp11
A	1		1		1		2
Pp12
..............
A	4		4		17		17
P..
B	1		1		1		1
Pp11
B	1		1		1		2
Pp12
..............
B	4		4		17		17
P..
C	1		1		1		1
Pp11
C	1		1		1		2
Pp12
..............
C	4		4		17		17
P..

When loading these files in either limma and limmaGUI only the A Field
is 
read. Not the values belonging to the B and C metagrid (which are the
replicates). 
Is there a simple way to solve this problem ? Or do I have to cut these
files into three parts ?

Secondly, using limma and the targets <- readTargets function you cannot
use the targets object directly in the read.imagene function. I solved
this to create matrix with ncol=2 and nrow same as in the targets
object. Then 
this can be used directly in read.imagene.
Next you would like to add color to your MA plots using controlTypes and
Spottypes. This doesn't work either because when reading the imagene
data files no columns RG$genes$Name and RG$genes$ID are created
(actually onlu RG$genes[['Gene ID']] exists.. I solved this by :
RG$genes$Name <- RG$genes[['Gene ID']] and same for ID. Then you will be
able to use the conrol types and colors for the colored MA plots. 
The same problem applies for limmaGUI. If you do not have a GAL file and
load the imagene files directly into limmaGUI you cannot assign colors
using spotTypes either. (Error messages appear already after loading the
files).

I hope that the developers can implement these suggestions/workarounds
for these problems ?

Yours, Bas van Breukelen



=================================
Dr. Ir. B. van Breukelen
PostDoc, Bioinformatics, Molecular genetics
Dept. of Biology. 
Room N407: H.R. Kruytgebouw
Padualaan 8
3574 CH Utrecht

Tel:       +31(0)30 253 3355
Mobile: +31(0)6 24 996046
e-mail:  b.vanbreukelen at bio.uu.nl
website: http://genomics.bio.uu.nl/

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