On Mon, Nov 10, 2008 at 10:42 AM, r.kandimalla <r.kandimalla@erasmusmc.nl>wrote:

> Dear all,
>
> I would like to here your comments and suggestions regarding my analysis
> described below.
> Im working with genomewide screening of CpG methylation with agilent two
> color CpG arrays with a common reference design.
> I have got the data of the future extraction and did loess normalisation,
> applied limma to check for the differentially regulated genes. In one of the
> comparision i have superficial and invasive tumors,
> surprisingly i saw zero differentially regulated genes which is quiet
> unrealistic.


Hi, Raju.

I didn't see any major problems with the code below, but someone with a
better eye might spot something.

What makes you say that no differentially-regulated genes is "quite
unrealistic"?  It is (unfortunately) quite possible for that to be the
case.  On the other hand, have you looked at basic quality control issues?
Sample relationships using unsupervised methods?  You mention that you are
using methylation arrays?  What have you done for normalization?  These are
all important questions that you should address thoroughly before you can
make final conclusions about your results.

Sean


>
> Here is the R session:
> >
> design1<-cbind(SM=c(1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0),
> I=c(0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1))
> > colnames(design1)<- c("SM", "I")
> > design1
> > dim(dataset1)
> [1] 237220     34
> > fit<- lmFit(dataset1, design1)
> > ngenes <- nrow (dataset1)
> > cont.matrix<- makeContrasts(SMvsI=SM-I, levels=design1)
> > cont.matrix
>      Contrasts
> Levels SMvsI
>    SM     1
>    I     -1
> > fit2<-contrasts.fit(fit, cont.matrix)
> > fit2 <-eBayes(fit2)
> > topTable(fit2, adjust="fdr")
> > SMvsI <- topTable(fit2, number=ngenes, adjust="fdr")
> > results <- decideTests(fit2,adjust.method="fdr",p.value=0.05)
> > summary(results)
>    SMvsI
> -1      0
> 0  237202
> 1       0
>
> Best regards,
>
> --
> Raju Kandimalla, PhD student
> Erasmus MC
> Department of Pathology
> JNI,Room H Be-302
> Dr. Molewaterplein 50
> 3015 GE Rotterdam-NL
> phone: +3110-7043093
> fax: +3110-7044762
> r.kandimalla@erasmusmc.nl
> http://www.erasmusmc.nl/pathologie
>
> _______________________________________________
> Bioconductor mailing list
> Bioconductor@stat.math.ethz.ch
> https://stat.ethz.ch/mailman/listinfo/bioconductor
> Search the archives:
> http://news.gmane.org/gmane.science.biology.informatics.conductor
>

	[[alternative HTML version deleted]]

