[BioC] limma help - choosing an approach

[john seers (IFR)]

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
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e-mail john.seers at bbsrc.ac.uk                         
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