Hi, After using SAM for a long time, I have started an "struggle" with the exceptional LIMMA package. After working with the example datasets and looking at the mailing list, I have begun to analyse our own data. In the simpler experiments, I have not found any trouble and the package did very well. But now I am trying to analyse a 2x2x2 factorial design, and I think I have run into problems, with my biologist background. We are using Affymetrix Genechip, studying an infection process in two different cell types and in two time points. There is two replicates for each point. The phenoData slot is: > pData(resRMA) Strain Infected Time Hyb01 A 1 6h Hyb02 A 1 6h Hyb03 A 1 24h Hyb04 A 1 24h Hyb05 A 0 6h Hyb06 A 0 6h Hyb07 A 0 24h Hyb08 A 0 24h Hyb09 B 1 6h Hyb10 B 1 6h Hyb11 B 1 24h Hyb12 B 1 24h Hyb13 B 0 6h Hyb14 B 0 6h Hyb15 B 0 24h Hyb16 B 0 24h I tried to create the following design matrix: > design<-model.matrix(~Strain*Infected*Time, data=pData(resRMA)) > design (Intercept) StrainB Infected1 Time6h StrainB:Infected1 StrainB:Time6h Infected1:Time6h StrainB:Infected1:Time6h Hyb01 1 0 1 1 0 0 1 0 Hyb02 1 0 1 1 0 0 1 0 Hyb03 1 0 1 0 0 0 0 0 Hyb04 1 0 1 0 0 0 0 0 Hyb05 1 0 0 1 0 0 0 0 Hyb06 1 0 0 1 0 0 0 0 Hyb07 1 0 0 0 0 0 0 0 Hyb08 1 0 0 0 0 0 0 0 Hyb09 1 1 1 1 1 1 1 1 Hyb10 1 1 1 1 1 1 1 1 Hyb11 1 1 1 0 1 0 0 0 Hyb12 1 1 1 0 1 0 0 0 Hyb13 1 1 0 1 0 1 0 0 Hyb14 1 1 0 1 0 1 0 0 Hyb15 1 1 0 0 0 0 0 0 Hyb16 1 1 0 0 0 0 0 0 attr(,"assign") [1] 0 1 2 3 4 5 6 7 attr(,"contrasts") attr(,"contrasts")$Strain [1] "contr.treatment" attr(,"contrasts")$Infected [1] "contr.treatment" attr(,"contrasts")$Time [1] "contr.treatment" Whick looked very logical for me, but very complicated (well, I was expecting something complex, anyway). So, before going into contrast analysis that could be meaningless, I decide to ask for some advice from Bioconductor´s list. First, is this model correct? Second, I am interested in several aspects (contrasts), which I can address if asked: For instance, differences between cell types without infection, and differences between cell types with infection (time excluded or included). Which contrasts can answer these questions? How many constrasts I can analyse? All of them? Is there sufficient degree of freedom? Thanks in advance for your assistance. Christian Probst Bioinformatics - IBMP [[alternative HTML version deleted]]

At 02:23 PM 1/04/2004, cmprobst wrote: >Hi, > >After using SAM for a long time, I have started an "struggle" with the >exceptional LIMMA package. > >After working with the example datasets and looking at the mailing list, I >have begun to analyse our own data. > >In the simpler experiments, I have not found any trouble and the package >did very well. > >But now I am trying to analyse a 2x2x2 factorial design, and I think I >have run into problems, with my biologist background. > >We are using Affymetrix Genechip, studying an infection process in two >different cell types and in two time points. There is two replicates for >each point. > >The phenoData slot is: > > > pData(resRMA) > Strain Infected Time >Hyb01 A 1 6h >Hyb02 A 1 6h >Hyb03 A 1 24h >Hyb04 A 1 24h >Hyb05 A 0 6h >Hyb06 A 0 6h >Hyb07 A 0 24h >Hyb08 A 0 24h >Hyb09 B 1 6h >Hyb10 B 1 6h >Hyb11 B 1 24h >Hyb12 B 1 24h >Hyb13 B 0 6h >Hyb14 B 0 6h >Hyb15 B 0 24h >Hyb16 B 0 24h > >I tried to create the following design matrix: > > > design<-model.matrix(~Strain*Infected*Time, data=pData(resRMA)) > > design > (Intercept) StrainB Infected1 Time6h StrainB:Infected1 > StrainB:Time6h Infected1:Time6h StrainB:Infected1:Time6h >Hyb01 1 0 1 1 0 >0 1 0 >Hyb02 1 0 1 1 0 >0 1 0 >Hyb03 1 0 1 0 0 >0 0 0 >Hyb04 1 0 1 0 0 >0 0 0 >Hyb05 1 0 0 1 0 >0 0 0 >Hyb06 1 0 0 1 0 >0 0 0 >Hyb07 1 0 0 0 0 >0 0 0 >Hyb08 1 0 0 0 0 >0 0 0 >Hyb09 1 1 1 1 1 >1 1 1 >Hyb10 1 1 1 1 1 >1 1 1 >Hyb11 1 1 1 0 1 >0 0 0 >Hyb12 1 1 1 0 1 >0 0 0 >Hyb13 1 1 0 1 0 >1 0 0 >Hyb14 1 1 0 1 0 >1 0 0 >Hyb15 1 1 0 0 0 >0 0 0 >Hyb16 1 1 0 0 0 >0 0 0 >attr(,"assign") >[1] 0 1 2 3 4 5 6 7 >attr(,"contrasts") >attr(,"contrasts")$Strain >[1] "contr.treatment" >attr(,"contrasts")$Infected >[1] "contr.treatment" >attr(,"contrasts")$Time >[1] "contr.treatment" > > >Whick looked very logical for me, but very complicated (well, I was >expecting something complex, anyway). > >So, before going into contrast analysis that could be meaningless, I >decide to ask for some advice from Bioconductor?s list. > >First, is this model correct? Assuming that your strains A and B are different cell types, rather than biological replicates of the same cell line, then this looks a correct model. >Second, I am interested in several aspects (contrasts), which I can >address if asked: > >For instance, differences between cell types without infection, and >differences between cell types with infection (time excluded or included). > >Which contrasts can answer these questions? Ah, this is the big question. I hope someone other than me will jump in here, because finding interpreting contrasts from factorial models is not specific to limma. > How many constrasts I can analyse? All of them? Yes. > Is there sufficient degree of freedom? Yes. Gordon >Thanks in advance for your assistance. > >Christian Probst >Bioinformatics - IBMP

>> At 02:23 PM 1/04/2004, cmprobst wrote: > > > >First, is this model correct? > > Assuming that your strains A and B are different cell types, rather than > biological replicates of the same cell line, then this looks a correct model. > Yes, Strain A and B are different cell types. There are two hybs for each 2x2x2 combination, in a total of 16 hybs (We are going to add another replicate). These two hybs are biological replicates, that could be included in the model, although I am not interested in them. I have not included them because I was afraid of doing a mistake. Perhaps, I could model like this, including the covariate "Batch": design<-model.matrix(~ Batch + Strain*Infected*Time, data=pData(resRMA) Anyway, thanks for your prompt answer, Gordon. [[alternative HTML version deleted]]