[BioC] read.maimages

[Jeannette [guest]]

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 ‘Quality assessment’ 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   

--
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