Agilent platform in microarray data analysis

Question

[nishimalhotra2612]

Hello all

I have been trying to do analysis of text files of agilent platform by using limma package from that I understood that we need target file which consist of file column

So my doubt is that do we have to create a target file and if we have to then what actually it should have what should be the contents of that file I m new to performing the analysis of agilent platform so trying to understand as much as I can but I m just confused regarding this

My data is of PC3 cell line

Geo accesion number

GSM3490386 GSM3490387

Thanks

Answer 1

[ATpoint]

Here is some minimal code snipped basically doing what the limma user guide suggests for these kinds of arrays, after downloading these text files for the PC3 cell line from the supplement (at the bottom) of https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE122997

I can already tell you that there are no DEGs at all. It is beyond me why people even do n=2 microarrays, that usually never has the power to reveal anything meaningful. You can additionally do plotMDS to see that the samples do not even cluster by treatment particularily well. Not sure what these data are good for.

library(limma)

files <- list.files("~/Downloads/GSE122997_RAW/", pattern=".txt", 
                    full.names=TRUE)

#/ read files
elistraw <- limma::read.maimages(files, source="agilent", green.only=TRUE)
#> Read /Users/atpoint/Downloads/GSE122997_RAW//GSM3490386_PC3_CONTROL_1.txt.gz 
#> Read /Users/atpoint/Downloads/GSE122997_RAW//GSM3490387_PC3_CONTROL_2.txt.gz 
#> Read /Users/atpoint/Downloads/GSE122997_RAW//GSM3490388_PC3_GSK126_1.txt.gz 
#> Read /Users/atpoint/Downloads/GSE122997_RAW//GSM3490389_PC3_GSK126_2.txt.gz 
#> Read /Users/atpoint/Downloads/GSE122997_RAW//GSM3490390_PC3_NPD13668_1.txt.gz 
#> Read /Users/atpoint/Downloads/GSE122997_RAW//GSM3490391_PC3_NPD13668_2.txt.gz
colnames(elistraw) <- gsub("GSM*_.", "", gsub(".txt.gz", "", basename(files)))

#/ normalize
elist <- normalizeBetweenArrays(elistraw, method="quantile")

#/ define targets
elist$targets$FileName # that is the order of the samples
#> [1] "/Users/atpoint/Downloads/GSE122997_RAW//GSM3490386_PC3_CONTROL_1.txt.gz" 
#> [2] "/Users/atpoint/Downloads/GSE122997_RAW//GSM3490387_PC3_CONTROL_2.txt.gz" 
#> [3] "/Users/atpoint/Downloads/GSE122997_RAW//GSM3490388_PC3_GSK126_1.txt.gz"  
#> [4] "/Users/atpoint/Downloads/GSE122997_RAW//GSM3490389_PC3_GSK126_2.txt.gz"  
#> [5] "/Users/atpoint/Downloads/GSE122997_RAW//GSM3490390_PC3_NPD13668_1.txt.gz"
#> [6] "/Users/atpoint/Downloads/GSE122997_RAW//GSM3490391_PC3_NPD13668_2.txt.gz"
elist$targets$group <- rep(c("control", "gsk126", "npd13668"), each=2)

#/ differential
design <- model.matrix(~group, elist$targets)
fit <- lmFit(elist, design)
fit <- eBayes(fit)

results_control_gsk126   <- topTable(fit, coef=2, number=Inf)
results_control_npd13668 <- topTable(fit, coef=3, number=Inf)

#/ no differential genes at all even at 25% FDR
summary(results_control_gsk126$adj.P.Val < .25)
#>    Mode   FALSE 
#> logical   62976
summary(results_control_npd13668$adj.P.Val < .25)
#>    Mode   FALSE 
#> logical   62976

[nishimalhotra2612]

Actually I have several samples of this cell line and I m using only untreated/control samples and I wanted to obtain the count file of all the samples and do the rankprod analysis

[nishimalhotra2612]

hi i tried to use the code but i m getting an error in using that

files <- list.files("C:/Users/User/OneDrive/Documents/agilent/GSE122997_RAW", pattern=".txt",full.names=TRUE)

elistraw <-read.maimages(files, source="agilent", green.only=TRUE)

but i m getting an error when i try to run the second one

Error in file(file, "r") : invalid 'description' argument

i searched about the error and find out that is occurs when you try to open multiple files but i m not understanding why it is happening

[nishimalhotra2612]

hi i solved that error actually i did not made the folder so it was my mistake now it works fine thanks for the code