[BioC] duplicated spots on oligonucleotide array

Leonardo Rocha leobernardesrocha at gmail.com
Tue Jun 26 17:17:48 CEST 2007

Dear List,

I am very sorry for the previous emails, I do not know what happened, so I 
trying to use another email. I am looking for help to account for 
duplication in analysis using lmFit in limma of data from a two-channel 
microarray. The experiment is comparing differences between breeds (A and N) 
using a dye-swap labelling. The array has the following layout:

[1] 12

[1] 4

[1] 19

[1] 19

[1] "PrintLayout"

 The array has been duplicated on the top half and the bottom half of the 
slide (spots on the tip 1 are duplicated on the tip 48, spots on the tip 2 
are duplicated on the tip 47, and so on). Moreover, the slide has control 
spots with different number of duplicates, which I have been attributed 
weights=0, so I am using only spots with 2 replicates ("genes"). I have been 
successful accounting for duplicated spots using the following commands:

 myfun <- function(x) as.numeric(x$Flags ==0)

 targets1 <-  as.matrix(read.table("targets.txt", header = TRUE)); targets1
     SlideNumber Name         FileName      Cy5      Cy3
 [1,] "1"         "Treatment1" "N16_A11.gpr" "A"  "N"
 [2,] "2"         "Treatment2" "A08_N15.gpr" "N" "A"
 [3,] "3"         "Treatment3" "N12_A06.gpr" "A"  "N"
[4,] "4"         "Treatment4" "A02_N07.gpr" "N" "A"

 filenames <- matrix (c(targets1[,3]),nrow=4,ncol=1); filenames
 [1,] "N16_A11.gpr"
[2,] "A08_N15.gpr"
[3,] "N12_A06.gpr"
[4,] "A02_N07.gpr"

 RG <- read.maimages(filenames, source="genepix", wt.fun=myfun)

 MA <- normalizeWithinArrays(RG, method="loess", bc.method="none")

 MA1 <-MA[order(RG$genes[,4]),]

 design = matrix(cbind(Dye = 1, c(-1,1, -1,1)), nrow=4, 
ncol=2,dimnames=list(c("N16_A11", "A08_N15", "N12_A06", "A02_N07"), 

 dupcor <-duplicateCorrelation(MA1, design, ndups=2, spacing=1)

 fit <- lmFit(MA1, design, ndups=2, spacing=1, 

 fit2 <- eBayes(fit)

 topTable (fit2, coef = "Treatment", adjust="BH", sort.by="P")

 However, if the duplicateCorrelation is used to estimate spatial 
correlation in the slide, I am not sure if makes sense to rearrange MA by 
GeneID and then apply the duplicateCorrelation with spacing=1, so I have 
tried to use (unsucessful) the argument spacing="topbottom".

 dupcor1 <-duplicateCorrelation(MA, design, ndups=2, spacing="topbottom")

 fit.topbottom <- lmFit(MA, design, ndups=2, spacing="topbottom", 
Error in nspots/ndups/spacing : non-numeric argument to binary operator

R version 2.5.0 (2007-04-23)

LC_COLLATE=English_United States.1252;LC_CTYPE=English_United 
States.1252;LC_NUMERIC=C;LC_TIME=English_United States.1252

 attached base packages:
[1] "stats"     "graphics"  "grDevices" "utils"     "datasets"  "methods" 

 other attached packages:
statmod    limma
 "1.3.0" "2.10.0"

 Is it correct what I am doing? Could anyone give me some suggestions with 
this problem?

 Thank you a lot for your help!


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