[BioC] limma help - choosing an approach

[Naomi Altman]

It looks OK to me except that

a) I needed to build my targets from a list of treatments for red and gree.
b) The design matrix will have more than 4 columns, as you have 3 
levels of concentration.  So there are 2 columns for concentration 
and 2 for time:concentration.

--Naomi

At 05:28 AM 9/13/2006, john seers \(IFR\) wrote:


>Hi Naomi
>
>Thanks again for taking the time to reply.
>
>Yes, your appreciation of the design is correct. (Not actually my design
>or I would understand it better!). The control is of concentration zero.
>
>By "single channel analysis" I think you mean the "Separate Channel
>Analysis" described in Chapter 9 of the limma user guide?
>
>So, this is what I have done. Does it make any sense?
>
>
>...... Read in data etc and normalise into MA.nba
>
>
># Read in the targets file and convert it to separate channel format
>targets<-readTargets(targetsfile)
>targetsC<-targetsA2C(targets)
>
># Make a design file
>Time<-targetsC$Time
>Conc<-targetsC$Conc
>Time<-factor(Time, levels=unique(Time))
>Conc<-factor(Conc, levels=unique(Conc))
>design<-model.matrix(~Time + Conc + Time:Conc)
>colnames(design)<-c("Intercept", "Time", "Conc", "TimeConc")
>
>#
>corfit<-intraspotCorrelation(MA.nba, design)
>fit<-lmscFit(MA.nba, design, correlation=corfit$consensus)
>
># contrast matrix
>contrast.matrix<-makeContrasts(Time, Conc, TimeConc,  levels=design)
>
># contrast timepoints and controls
>fit2<- contrasts.fit(fit, contrast.matrix)
>
>
># eBayes
>eb<- eBayes(fit2)
>
>
>ngenes<-20
>topa1<-topTable(eb, coef=1, number=ngenes, adjust="none", sort.by="M")
>topa2<-topTable(eb, coef=2, number=ngenes, adjust="none", sort.by="M")
>topa3<-topTable(eb, coef=3, number=ngenes, adjust="none", sort.by="M")
>
>Thanks.
>
>
>John Seers
>
>
>Target file:
>
>SlideNumber     FileName        Cy3     Cy5     Time    Conc
>598     598new.gpr      f100t1  Control t1      c100
>599     599new.gpr      f20t4   Control t4      c20
>600     600new.gpr      f100t4  Control t1      c100
>617     617new.gpr      f20t1   Control t1      c20
>621     621new.gpr      f20t1   Control t1      c20
>637     637new.gpr      f20t4   Control t4      c20
>638     638new.gpr      f20t1   Control t1      c20
>639     639new.gpr      f100t1  Control t1      c100
>748     748new.gpr      f20t4   Control t4      c20
>751     751new.gpr      f20t4   Control t4      c20
>833     833new.gpr      f100t4  Control t4      c100
>835     835new.gpr      f100t1  Control t1      c100
>836     836new.gpr      f100t4  Control t4      c100
>957     957new.gpr      f100t1  Control t1      c100
>958     958new.gpr      f20t1   Control t1      c20
>
>
>
>---
>
>John Seers
>Institute of Food Research
>Norwich Research Park
>Colney
>Norwich
>NR4 7UA
>
>
>tel +44 (0)1603 251497
>fax +44 (0)1603 507723
>e-mail john.seers at bbsrc.ac.uk
>e-disclaimer at http://www.ifr.ac.uk/edisclaimer/
>
>Web sites:
>
>www.ifr.ac.uk
>www.foodandhealthnetwork.com
>
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Naomi S. Altman                                814-865-3791 (voice)
Associate Professor
Dept. of Statistics                              814-863-7114 (fax)
Penn State University                         814-865-1348 (Statistics)
University Park, PA 16802-2111