[BioC] basic limma questions

[Endre Sebestyen]

Thanks for your answer. A little more details :

I had a control and a treatment, with 3 technical replicates and no
dye-swaps. Cy3 was the treated, Cy5 the untreated.

The ArrayVision result file looks like this :

                                 Ctrl    Ctrl    Ctrl    Data    Data
  Data
Spot labels     VOL - MDC       Bkgd    sVOL    VOL - MDC       Bkgd
 sVOL    Ratio (sVOL):  Data / Ctrl      Diff (sVOL):  Data - Ctrl
MZ00023554 - TC248295 (1)       87573.5998      29969.817
57603.783       51217.7619      15349.622       35868.140       0.623
 -21735.643
MZ00023408 - TC248006 (1)       29389.2252      29831.153       0.000
 15896.2700      15349.622       546.648 1.0000e+100     546.648

I extracted the ID, Ctrl VOL, Ctrl Bkgd, Data VOL, Data Bkgd columns.
When I used the read.maimages function, Ctrl VOL became R, Data VOL
became G, Ctrl Bkgd became Rb, DataBkgd became Gb.

Endre


On Mon, Mar 31, 2008 at 5:36 AM, Gordon K Smyth <smyth at wehi.edu.au> wrote:
> Dear Endre,
>
>  I suspect the reason you didn't get a rsponse to your original posting is
>  that your questions are not very specific.
>
>  Assuming that you have three replicate arrays comparing a control to a
>  treatment, your analysis looks fine.
>
>  I can't give you any response to the problem reading ArrayVision format
>  because you haven't shown us the error you received or the structure of
>  your data files.  Therefore we can't see if your files really are in
>  ArrayVision format or diagnose what the read problem is.
>
>  I can't tell you if the column names are correct, because ArrayVision
>  columns are used-defined.  Your interpretation appears reasonable.
>
>  You ask if your design is correct, but you don't give any information
>  about your experiment, eg the targets information.  I have to assume that
>  you have three replicate arrays.
>
>  In my opinion, there is no good way to combine irregular numbers of
>  within-array gene replicates, especially if the probes are not indentical.
>
>  Hope this helps
>  Gordon
>
>  > Date: Fri, 28 Mar 2008 12:51:39 +0100
>  > From: "Endre Sebestyen" <endre.sebestyen at gmail.com>
>  > Subject: [BioC] basic limma questions
>  > To: Bioconductor <bioconductor at stat.math.ethz.ch>
>  >
>  > Again :
>  >
>  > Hi!
>  >
>  > I'm a beginner in limma and bioconductor, and I'd like to ask a few
>  > basic questions.
>  >
>  > I wrote the following script :
>  >
>  > library(limma)
>  > targets <- readTargets("Targets1.txt")
>  > RG <- read.maimages(targets$FileName, columns=list(R="CtrlVol",
>  > G="DataVol", Rb="CtrlBg", Gb="DataBg"),
>  > annotation=c("ID","Name","Rep"))
>  > RG$genes <- readGAL("maize.gal")
>  > RG$printer <- getLayout(RG$genes)
>  > spottypes <- readSpotTypes()
>  > RG$genes$Status <- controlStatus(spottypes, RG)
>  > bgCorr <- backgroundCorrect(RG, method="movingmin")
>  > nWithin <-normalizeWithinArrays(bgCorr, method="loess")
>  > nBetween <- normalizeBetweenArrays(nWithin, method="Aquantile")
>  > design <- c(1,1,1)
>  > isGene <- nBetween$genes$Status == "cDNA"
>  > fit <- lmFit(nBetween[isGene, ], design)
>  > fit <- eBayes(fit)
>  > res <- topTable(fit, number=1000)
>  > write.table(res, file="results24top1000.txt", sep="+++")
>  >
>  > First, limma didn't recognize the ArrayVision format, and I had to
>  > parse the raw data and define the columns myself. Is it correct to
>  > pass the CtrlVol to R, DataVol to G, etc? The other question is that
>  > I'm not sure about the design. Cy3 was the treated, Cy5 the control,
>  > but after I used the read.maimages function and defined the values
>  > myself, this design should be OK. Am I right?
>  >
>  > Last question : how can I combine the replicates on a chip? I have
>  > some genes with 2,3,etc replicates, but not all. This is the 46k maize
>  > array from www.maizearray.org
>  >
>  > Thanks for any comment and help.
>  >
>  >
>  > Endre Sebestyen
>  >
>  > --
>  > Agricultural Research Institute of the Hungarian Academy of Sciences
>  > Department of Applied Genomics
>  > H-2462 Martonv?s?r, Brunszvik street 2.
>



-- 
Agricultural Research Institute of the Hungarian Academy of Sciences
Department of Applied Genomics
H-2462 Martonvásár, Brunszvik street 2.