Dear Jeannette,

GSE37326 is using standard Agilent arrays, so you could set
the source argument to read.maimages to "agilent" and it should
find the right columns from your text files. Otherwise see the
annotation argument in ?read.maimages in order to get gene
annotation.

You should certainly not use fit2 in your call to topTable() if you
have used fit for all of your analysis :-) and you can set
number = Inf
to include all features in your output table.

For QA please see the very helpful limma user's guide and package
arrayQualityMetrics.

Generally, I'd try to include *all* arrays in your limma analysis
instead of only the three you're interested in since that should
improve your power -- but I just see that GSE37326 is actually
using two different array platforms and that may complicate things... :-(

Hope that helps,

 - axel


On Tue, Jul 15, 2014 at 6:20 PM, Jeannette [guest] <guest@bioconductor.org>
wrote:

> I am an absolut beginner in R and limma.
> I would like to analyze 2 colour microarray data (GSE37326):
> normalization, quality control, estimate diff. genes
>
> I am only interested in finding diff. genes for one substance (slides:
> GSM915419_NP_1, GSM915420_NP_2 and GSM915421_NP_3).
>
> targets.txt
> SampleNumber    FileName        Cy3     Cy5
> 1       GSM915419_NP_1.txt      DMSO    NP
> 2       GSM915420_NP_2.txt      DMSO    NP
> 3       GSM915421_NP_3.txt      DMSO    NP
>
>
> Is this correct for my files?
> > targets <- readTargets("targets_test_.txt")
> > RG <- read.maimages(targets, columns=list(R="F635 Median",G="F532
> Median",Rb="B635",Gb="B532"))
> > MA <- normalizeWithinArrays(RG, method="loess")
> > fit <- lmFit(MA)
> > fit <- eBayes(fit)
> > output <- topTable(fit2, adjust="BH", coef="NP", number=100000)
> > write.table(output, file="NP_vs_DMSO_15072014.txt", sep="t", quote=FALSE)
>
> I am really not sure if the output table is correct (especially my use of
> read.maimages?).
> How to perform a â€˜Quality assessmentâ€™ before and after normalization?
> How to get gene annotation?
>
> Any feedback is very welcome.
> Thanks and best regards,
> Jeannette
>
>
>  -- output of sessionInfo():
>
> R version 3.0.2 (2013-09-25)
> Platform: x86_64-w64-mingw32/x64 (64-bit)
>
> locale:
> [1] LC_COLLATE=German_Germany.1252  LC_CTYPE=German_Germany.1252
> [3] LC_MONETARY=German_Germany.1252 LC_NUMERIC=C
> [5] LC_TIME=German_Germany.1252
>
> attached base packages:
> [1] parallel  stats     graphics  grDevices utils     datasets  methods
> base
>
> other attached packages:
> [1] limma_3.18.13      affy_1.40.0        Biobase_2.22.0
> BiocGenerics_0.8.0
>
> loaded via a namespace (and not attached):
> [1] affyio_1.30.0         BiocInstaller_1.12.1  preprocessCore_1.24.0
> [4] tools_3.0.2           zlibbioc_1.8.0
> R version 3.0.2 (2013-09-25)
> Platform: x86_64-w64-mingw32/x64 (64-bit)
>
> locale:
> [1] LC_COLLATE=German_Germany.1252  LC_CTYPE=German_Germany.1252
> [3] LC_MONETARY=German_Germany.1252 LC_NUMERIC=C
> [5] LC_TIME=German_Germany.1252
>
> attached base packages:
> [1] parallel  stats     graphics  grDevices utils     datasets  methods
> base
>
> other attached packages:
> [1] limma_3.18.13      affy_1.40.0        Biobase_2.22.0
> BiocGenerics_0.8.0
>
> loaded via a namespace (and not attached):
> [1] affyio_1.30.0         BiocInstaller_1.12.1  preprocessCore_1.24.0
> [4] tools_3.0.2           zlibbioc_1.8.0
>
> --
> Sent via the guest posting facility at bioconductor.org.
>
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>



-- 


Axel Klenk
Research Informatician
Information Management Drug Discovery

Actelion Pharmaceuticals Ltd. • Gewerbestrasse 16 • CH-4123 Allschwil •
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