Single channel spike in controls with custom microRNA slides - Normalization help needed
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David ▴ 860
@david-3335
Last seen 3.3 years ago
Hi, I'm working with custom slides(Cy5) and working in the normalization of the arrays. I have three arrays (technical replicates). I have sucesfully normalized the data using vsn, however i would like to normalize using spike in controls. My controls are annotated as CTL-1 to x and i would like to do etiher a normalization by block per array or the mean of all the controls per array. The gal file is also loaded with all the array structure. Each block contains spike in controls. Here is the code: library(limma) library(RColorBrewer) library(vsn) Cy5 <- "F635 Mean" Cy5b <- "B635 Mean" targets <- readTargets("targets.txt") #My gpr files do only contain 1 channel (Cy5) RG <- read.maimages( targets$FileName,source="genepix",columns=list(R=Cy5,G=Cy5, Rb=Cy5b, Gb=Cy5b)) RG$G <- NULL RG$Gb <- NULL RG$genes <- readGAL("array_human_mirs.gal") #Here are my spike in controls for normalization isSpikeIn <- grep("CTL", RG$genes$Name) #The vsn normalization works fine mat <- vsnMatrix(RG$R) However i would like to normaliza using my spikein controls by block or by using the mean of all controls. Could you help on that ?? thanks, david [[alternative HTML version deleted]]
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Entering edit mode
David ▴ 860
@david-3335
Last seen 3.3 years ago
Hi, I'm working with custom slides(Cy5) and working in the normalization of the arrays. I have three arrays (technical replicates). I have sucesfully normalized the data using vsn, however i would like to normalize using spike in controls. My controls are annotated as CTL-1 to x and i would like to do etiher a normalization by block per array or the mean of all the controls per array. The gal file is also loaded with all the array structure. Each block contains spike in controls. Here is the code: library(limma) library(RColorBrewer) library(vsn) Cy5 <- "F635 Mean" Cy5b <- "B635 Mean" targets <- readTargets("targets.txt") #My gpr files do only contain 1 channel (Cy5) RG <- read.maimages( targets$FileName,source="genepix",columns=list(R=Cy5,G=Cy5, Rb=Cy5b, Gb=Cy5b)) RG$G <- NULL RG$Gb <- NULL RG$genes <- readGAL("array_human_mirs.gal") #Here are my spike in controls for normalization isSpikeIn <- grep("CTL", RG$genes$Name) #The vsn normalization works fine mat <- vsnMatrix(RG$R) However i would like to normaliza using my spikein controls by block or by using the mean of all controls. Could you help on that ?? thanks, david [[alternative HTML version deleted]]
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