[BioC] Problems in limma package

[Binita Dutta]

Dear All,

I have done cDNA microarray (dye swap) experiments  (two repeats)  with 
samples from wild type and mutant  mice(experiment is very similar to Swirl 
experiment given in tutorials). I tried normalising data with "limma" 
package with following commands:
library(limma)
Warning message:
package limma was built under R version 1.8.1
RG<-read.maimages(files=c("binita1.txt","binita2.txt","binita3.txt","binita4.txt"), 
columns=list(Rf = "CH1_NBC_INT", Rb = "CH1_SPOT_BKGD", Gf = "CH2_NBC_INT", 
Gb = "CH2_SPOT_BKGD", Name="GENE_DESCRIPTION",verbose=TRUE,sep=" 
\t",  quote = "\"'", dec = "."))
  RG$genes<-read.table("binitaSample2.txt", sep="\t",header=TRUE,  quote = 
"\"'",fill=TRUE)
layout=list(ngrid.r=2,ngrid.c=12,nspot.r=45,nspot.c=21)
  RG$printer<-layout
  RG<-backgroundCorrect(RG, method="minimum")
  MA<-normalizeWithinArrays(RG,layout=RG$printer)
plotMA(MA)
As expected, I get MA plot

However, when i try normalising  the data between the arrays with following 
commands:
MA<-normalizeBetweenArrays(MA)
  plotMA(MA)
The graph flips  (if you want i can send graphs)

i.e X axis is reversed

design<-c(-1,1,-1,1)

fit<-lmFit(MA,design)
fit<-eBayes(fit)
  qqt(fit$t,df=fit$df.prior+fit$df.residual,pch=16,cex=0.1)
top<-topTable(fit,number=22680,adjust="fdr")
ord<-order(fit$lods,decreasing=TRUE)
top30<-ord[1:30]
plot(A,M,pch=16,cex=0.1)
text(A[top30],M[top30],labels=MA$genes[top30,"SPOT.LABEL"],cex=0.8,col="blue")
I get following errors:

1) P.Values which i obtain is 1 or above 1, i tried adjusting P.Value with 
"Holms" etc but get the same result. However, the same experiment when i 
analyse thorugh other progams, the P.Values are very less (less than 0.001) 
for differentially expressed genes.
3) i have problems in subseting topTables also.
subset<-subset(topTable,P.Value<0.01,select=MA$genes)
Error in subset.default(topTable, P.Value < 0.01, select = MA$genes) :
         Object "P.Value" not found
2)on MA plot i want to highlight the top 30 genens with "SPOT LABEL" which 
is there on MA$genes, but the program picks up  some number which does not 
corresonds to the SPOT.LABEL.
I have shown here, top 10 genes
SPOT.LABEL CIU CLONE.ID     M         A     t P.Value     B
4459        4459 MM6    25179  1.91  1.627766 11.18       1 -2.82
14466      14466 MM6    14674  1.62  0.695737 10.35       1 -2.84
17413      17413 MM6    17720  2.48 -1.036538 10.08       1 -2.85
21140      21140 MM6    16316  1.57  0.000431  9.80       1 -2.85
18070      18070 MM6     9920  1.61  1.888210  9.61       1 -2.86
776          776 MM6    27323  1.72  1.873411  9.45       1 -2.86
12014      12014 MM6    25084  1.74  1.226841  9.43       1 -2.87
19423      19423 MM6    20962 -1.44  4.009106 -9.22       1 -2.87
17258      17258 MM6    13529  2.31  0.289187  9.06       1 -2.88
21565      21565 MM6    27496  1.60  1.794250  8.78       1 -2.89

Help in this regards will be highly appreciated.
Sincerely yours,
Binita

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Binita Dutta, PhD
MicroArray Facility(MAF)
UZ Gasthuisberg
Onderwijs en Navorsing
Herestraat 49
3000 Leuven
Belgium