Re: Any problem with the design matrix or the contrasts? Thanks

Hi, Ryan

Thanks a lot for your comments! It is good to know that testing for a main effect using only the additive model may not be valid in the presence of an interaction effect, which I agree as I recall that from my statistics course. I did what you suggested and tested for the interaction effect in the interaction model as below. There are no sig diff genes in the end (no interaction effect). So I think now it is safe to apply the additive model. But what if there is an interaction effect? In that case, to test for ARpos vs. ARneg, I think the model should be y=b0 + b1*gender + b2*AR + b3*chip + b3*gender*AR , so the code would be: design <- model.matrix(~0+AR+gender+chip+AR*gender), right ??  In addition, I guess there is no difference in testing for the 2nd question Mpos vs. Fpos using the in
 teraction model, right ?? 

Treat <- factor(paste(targets$gender,targets$AR,sep="."))
design <- model.matrix(~0+Treat+chip)
cm <- makeContrasts((F.pos-M.pos)-(F.neg-M.neg),levels=design)

Thanks,
Xiayu




-----Original Message-----
From: Ryan C. Thompson [mailto:[email protected]] 
Sent: Friday, August 08, 2014 4:14 PM
To: Rao,Xiayu; 'bioconductor-J/1JLT8/[email protected]'
Subject: Re: [BioC] Any problem with the design matrix or the contrasts? Thanks

Hi,

You're going to need to address your two scientific questions using separate fits. This is because your first question (pos vs neg) is testing a main effect, and you are effectively fitting an interaction model (one coefficient for each combination of AR & gender). So your interaction model is correct for testing the second question, which is testing one factor within only a single level of the other. But for testing the main effect, you'll need to fit an additive model using the formula suggested by Federico. Furthermore, the test for a main effect may not be valid in the presence of an interaction effect, so you should test for that too using the interaction model, and then look closely at the data before trusting probe sets that are significant for both the main effect and interaction e
 ffect. The interaction contrast using your design would be ((F.pos-M.pos)-(F.neg-M.neg)).

-Ryan

On 08/01/2014 08:07 AM, Rao,Xiayu wrote:
> Hello,
>
> I learned from posts in the forum and analyzed my data using the suggested design and contrasts. It turns out there are no sig genes or few in the end. Could you please help check if the design does not fit the data or the contrasts made are incorrect??
>
> #the research questions are 1) to compare between pos and neg for AR,  and 2) Male.pos vs. Female.pos.
>
> Treat <- factor(paste(targets$gender,targets$AR,sep="."))
> chip <- factor(targets$chip)
> design <- model.matrix(~0+Treat+chip)
> colnames(design)[1:4] <- levels(Treat) fit <- lmFit(y,design)
>
> cm <- makeContrasts(posVSneg=(F.pos+M.pos-F.neg-M.neg)/2, 
> MposVSFpos=M.pos-F.pos, levels=design)
> fit2 <- contrasts.fit(fit,cm)
> fit2 <- eBayes(fit2)
> topTable(fit2, coef="posVSneg", sort.by="p")           #0 sig genes
> topTable(fit2, n=20,coef="MposVSFpos", sort.by="p")             #4 sig genes
>
> targets.txt
> sample gender AR          chip
> s1            F              pos         1
> s2            F              neg        1
> s3            M            neg        2
> s4            M            pos         2
> s5            F              neg        2
> s6            M            pos         2
> s7            M            pos         2
> s8            M            pos         3
> s9            M            pos         3
> s10         M            pos         3
> s11         M            pos         3
> s12         F              pos         4
> s13         F              pos         4
> s14         M            pos         4
> s15         M            pos         4
> s16         F              pos         5
> s17         M            pos         5
> s18         M            pos         5
> s19         M            neg        6
> s20         M            neg        6
> s21         F              neg        6
> s22         F              pos         6
> s23         F              pos         6
> s24         F              neg        6
> s25         F              pos         6
> s26         F              pos         6
> s27         F              pos         6
> s28         M            neg        6
>
>
> Thanks,
> Xiayu
>
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