gene expression with .txt file

Question

[Joe Kherery]

Hello everyone,

I have a text file containing the gene expression values of 5 control samples and 5 treated with a drug.

MY DATA

Above, as my data is arranged in .txt, but when I see a boxplot of this data they don't seem normal to me.

Is there any way to normalize the data?

or could someone measure a light on how to process this data?

MY BOX PLOT.

MY CODE

library(limma)

library (Biobase)

eset <- readExpressionSet("Data.txt", header=T)

View(eset)

targets <- read.delim("Target.txt", header=TRUE)

 View(targets)

f <- factor(targets$Target, levels = unique(targets$Target))

design <- model.matrix(~0 + f)

colnames(design) <- levels(f)

f <- factor(targets$Target, levels = unique(targets$Target))

design <- model.matrix(~0 + f)

colnames(design) <- levels(f)

fit <- lmFit(eset, design)

write.table(fit, file="fit.txt", sep="\t", quote=FALSE)

contrast.matrix <- makeContrasts("Drug-Control", levels=design)

fit2 <- contrasts.fit(fit, contrast.matrix)

fit2 <- eBayes(fit2)

output <- topTreat(fit2, coef=1, number=Inf, adjust.method="BH")

write.table(output, file="Drug-Control_1.txt", sep="\t", quote=FALSE)

[Kevin Blighe]

What is the source of the data? This is important to know.

[Joe Kherery]

Hey Kevin,

Unfortunately I don't have any more information about the data, platform or how it was generated, I just havethe data.txt like the image and I need the differentially expressed genes between drug-control.

Is there anything I can do?

[Kevin Blighe]

Oh really? - that is a bit strange. Does the person who gave it to you not know the source?

You could do some rudimentary quantile normalisation, which should help.

[Joe Kherery]

Yes, really... He only knows that they are from cancer cells, treated with a drug. I requested the raw data, but he said they didn't have it.

Could you give me an example of how to do that quantile normalization?

[Kevin Blighe]

Hey, there is a function from the preprocessCore package that can do it for you, if you take a look at the final piece in this blog posting: https://davetang.org/muse/2014/07/07/quantile-normalisation-in-r/

I mean, quantile normalisation is just one part of the RMA normalisation process, which goes:

• background correction
• quantile normalisation
• log [base 2] transformation

So, the assumption that you'd be making here is that background correction has already been performed.

Apart from anything else, you may want to find out how there is data lying around and nobody knows the source!

[Joe Kherery]

Dear Kevin,

Thank you so much for your reply.

I will follow your recommendations.

[Joe Kherery]

when i try to normalize.quantiles (eset) I always get the error

Matrix expected in normalize.quantiles

[Kevin Blighe]

Ah, you may have to coerce it to a data matrix via data.matrix().

How many genes are in it, by the way?

[Joe Kherery]

hello again,

I think there are about 20042