Question: gene expression with .txt file
0
gravatar for Joe Kherery
9 months ago by
Joe Kherery100
Joe Kherery100 wrote:

Hello everyone,

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

MY DATA

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.

barplot

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)
txt R gene • 528 views
ADD COMMENTlink modified 9 months ago • written 9 months ago by Joe Kherery100
2

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

ADD REPLYlink written 9 months ago by Kevin Blighe63k

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?

ADD REPLYlink written 9 months ago by Joe Kherery100
1

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.

ADD REPLYlink written 9 months ago by Kevin Blighe63k

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?

ADD REPLYlink written 9 months ago by Joe Kherery100
1

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!

ADD REPLYlink modified 9 months ago • written 9 months ago by Kevin Blighe63k

Dear Kevin,

Thank you so much for your reply.

I will follow your recommendations.

ADD REPLYlink written 9 months ago by Joe Kherery100

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

Matrix expected in normalize.quantiles

ADD REPLYlink written 9 months ago by Joe Kherery100
1

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

How many genes are in it, by the way?

ADD REPLYlink written 9 months ago by Kevin Blighe63k

hello again,

I think there are about 20042

ADD REPLYlink written 9 months ago by Joe Kherery100
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