[BioC] Limma analysis

Pete p.underhill at har.mrc.ac.uk
Thu Oct 30 04:25:31 MET 2003

Sorry I didn't explain that particularly well, firstly how do I create a
design matrix for this experiment?
Also as far as I can see the read.imagene function doesn't read in the flag
information for each file, in imagene each spot can have a flag value from
0-8? and in this case we want to ignore completely anything which is non
zero. Presumably this could be specified in the wt.fun argument, but i'm
unsure precisely how to do this? I have tried to modify the wtflags function
but without success.


----- Original Message -----
From: "Gordon Smyth" <smyth at wehi.edu.au>
To: "Pete" <p.underhill at har.mrc.ac.uk>
Cc: <bioconductor at stat.math.ethz.ch>
Sent: Tuesday, October 28, 2003 2:50 AM
Subject: Re: [BioC] Limma analysis

> At 10:04 AM 28/10/2003, Pete wrote:
> >Hi all,
> >I have been using limma now for a couple of weeks, and I think I have
> >much got the hang of most of it. However, now I want to analyse a
> >more complex experiment, can anyone give me some guidance as how to deal
> >with this.
> >      Firstly the experimental design is as follows, there are four
> >wildtype tissue A, wildtype tissue B, mutant tissue A and mutant tissue
> >Each sample has been compared to eachother in triplicate (inlcuding a dye
> >swap, and one independant sample). To complicate things further an
> >additional set of WT A v WT B was also done in triplicate using a
> >method.
> >     The slides are 7.5k oligos spotted in duplicate ( the duplicates are
> >the same block 10 rows below the first copy), although there are control
> >genes which appear more than twice on the arrays. My files are imagene
> >output files where the cy5 and cy3 are contained in separate files.  Also
> >the imagene output contains spots which are flagged and would need to be
> >removed from the analysis (meaning that a particular gene could have none
> >one or two measurements for it).
> >
> >What do you think the best strategy to deal with this design is?
> Well, everything in your experiment is straight down the line as far as
> limma is concerned. You haven't really said what is is about this
> experiment which you're not sure how to deal with. Is the problem the
> design matrix or something else?
> Gordon
> >Cheers
> >
> >Pete
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