[BioC] Design question in LIMMA

[Gordon K Smyth]

Dear Nataliya,

> Date: Fri, 11 Nov 2005 01:09:48 +0100
> From: Nataliya Yeremenko <eremenko at science.uva.nl>
> Subject: [BioC] Design question in LIMMA
> To: Bioconductor List <bioconductor at stat.math.ethz.ch>
>
> Hello
> This is a long letter about my efforts of analysis of data in Limma.
>
> I have a question about the proper design of my experiment
> I have 3 groups to compare: A, O, and Y.
> With 5 A, 8 O, and 7 Y biological samples.
> I've performed in total 28 two-color microarrays (Agilent 44K)
> with a mixed number of dye-swaps.
>
> My targets file is:
> HybID    fileName    sampleID    Cy3    Cy5
> 1    124879.txt    YvsO    Y1    O1
> 2    124880.txt    OvsY    O1    Y1
> 3    124919.txt    YvsO    Y2    O2
> 4    124972.txt    OvsY    O2    Y2
> 5    124984.txt    YvsO    Y3    O3
> 6    124957.txt    OvsY    O3    Y3
> 7    130365.txt    YvsO    Y4    O4
> 8    130366.txt    OvsY    O4    Y4
> 9    130372.txt    YvsO    Y5    O5
> 10    130374.txt    OvsY    O5    Y5
> 11    124881.txt    AvsO    A1    O1
> 12    124882.txt    OvsA    O1    A1
> 13    124982.txt    AvsO    A2    O2
> 14    124983.txt    OvsA    O2    A2
> 15    130351.txt    AvsO    A2    O2
> 16    124985.txt    AvsO    A3    O3
> 17    124958.txt    OvsA    O3    A3
> 18    130352.txt    AvsO    A3    O3
> 21    130355.txt    AvsO    A4    O4
> 22    130361.txt    OvsA    O4    A4
> 23    130362.txt    AvsO    A5    O5
> 24    130363.txt    OvsA    O5    A5
> 19    130353.txt    AvsO    A6    O6
> 20    130354.txt    OvsA    O6    A6
> 25    130375.txt    AvsO    A7    O7
> 26    130376.txt    OvsA    O8    A7
> 27    130377.txt    AvsO    A7    O6
> 28    130396.txt    OvsA    O6    A7
>
> After import of the data, normalization within and between arrays and
> evaluation of diagnostic plots, the question about fitting linear model
> arised.
>
> I didn't succeed to create proper direct design for all 3 groups.
> However for separate Y vs O, and A vs O it works Ok
> with the design of type:
> design <- cbind(Y1vsO1 = c(-1,1,0,0,0,0,0,0,0,0),
>                 Y2vsO2 = c(0,0,-1,1,0,0,0,0,0,0),
>                 Y3vsO3 = c(0,0,0,0,-1,1,0,0,0,0),
>                 Y4vsO4 = c(0,0,0,0,0,0,-1,1,0,0),
>                 Y5vsO5 = c(0,0,0,0,0,0,0,0,-1,1))
> But here I think I loose info about O6, O7 and O8 which are extra
> biological replicates.
> The same is valid for A vs O and I had to exclude last three hybs.
>
> What is your advise in that case?
>
> I have also tried to split all data into separate channels,
> so producing 56 single-channel data sets.
> (The reason for that was that I have even and odd number of replicates
> for my groups mixed in hybridizations)
>  >targets2 <- targetsA2C(targets)
>  >u <- unique(targets2$Target)
>  >f <- factor(targets2$Target, levels=u)
>  >design <- model.matrix(~0+f)
>  >colnames(design) <- u
>
> It works not bad until
>  >corfit <- intraspotCorrelation(MA, design)
> It took a lot of time and generated 43 warnings: "exceed amount of
> iterations ...."

A number of warnings like this are not a problem.  BTW, please don't put text in quotes unless it
really is an exact quotation.  I would never word a warning message in that way.

> fit <- lmscFit(MA, design, correlation=corfit$consensus)
>
> Than a BIG question appeared: "What is the contrasts matrix is in my case?"
> cont.matrix <-
> makeContrasts("(A1+A2+A3+...)/7-(O1+O2+O3+...)/8",levels=design)
> fit2 <- contrasts.fit(fit, cont.matrix)
> fit2 <- eBayes(fit2)
> topTable(fit2, adjust="BH", number=30, resort.by"M")
>
> Is it correct for A vs O comparison?
> I've got the table finally...
> And needles to say top 10 is different from my direct design A vs O (see
> above)

This seems about the best you can do with this experiment in limma.

This experiment has a complex design and you should probably consult a local statistician on it,
say Prof van Houwelingen or Dr te Meerman.

Best wishes
Gordon

> Regards
> Nataliya
>
> --
> Dr. Nataliya Yeremenko
>
> Universiteit van Amsterdam
> Faculty of Science
> IBED/AMB (Aquatische Microbiologie)
> Nieuwe Achtergracht 127
> NL-1018WS Amsterdam
> the Netherlands

> tel. + 31 20 5257089
> fax  + 31 20 5257064