Question: RNA Seq Analysis in R
0
gravatar for anasjamshed1994
3 months ago by
anasjamshed199420 wrote:

I choose microarray data GSE75693 of 30 patients with stable kidney transplantation and 15 with BKVN to identify differentially expressed genes (DEGs). I performed this in GEO2R and find R script there and Runs R script Successfully on R studio as well. The R script is :

#   Differential expression analysis with limma
library(Biobase)
library(GEOquery)
library(limma)

# load series and platform data from GEO

gset <- getGEO("GSE75693", GSEMatrix =TRUE, AnnotGPL=TRUE)
if (length(gset) > 1) idx <- grep("GPL570", attr(gset, "names")) else idx <- 1
gset <- gset[[idx]]

# make proper column names to match toptable 
fvarLabels(gset) <- make.names(fvarLabels(gset))

# group names for all samples
gsms <- paste0("000000000000000000000000000000XXXXXXXXXXXXXXX11111",
        "1111111111XXXXXXXXXXXXXXXXXXX")
sml <- c()
for (i in 1:nchar(gsms)) { sml[i] <- substr(gsms,i,i) }

# eliminate samples marked as "X"
sel <- which(sml != "X")
sml <- sml[sel]
gset <- gset[ ,sel]

# log2 transform
exprs(gset) <- log2(exprs(gset))

# set up the data and proceed with analysis
sml <- paste("G", sml, sep="")    # set group names
fl <- as.factor(sml)
gset$description <- fl
design <- model.matrix(~ description + 0, gset)
colnames(design) <- levels(fl)
fit <- lmFit(gset, design)
cont.matrix <- makeContrasts(G1-G0, levels=design)
fit2 <- contrasts.fit(fit, cont.matrix)
fit2 <- eBayes(fit2, 0.01)
tT <- topTable(fit2, adjust="fdr", sort.by="B", number=1250)

tT <- subset(tT, select=c("ID","adj.P.Val","P.Value","t","B","logFC","Gene.symbol","Gene.title"))
write.table(tT, file=stdout(), row.names=F, sep="\t")

But I want to select DEGs based on the threshold P < 0.01 and fold change >2.0. How can I do this through R script?

rna-seq microarray degs R • 376 views
ADD COMMENTlink modified 3 months ago by Kevin Blighe67k • written 3 months ago by anasjamshed199420

Hello anasjamshed1994!

It appears that your post has been cross-posted to another site: https://bioinformatics.stackexchange.com/questions/14070/deg-analysis-in-r-through-bioconductor

This is typically not recommended as it runs the risk of annoying people in both communities.

ADD REPLYlink written 3 months ago by RamRS30k

Your cross-posting on a different site, especially after someone has already started helping you here, is disrespectful and unprofessional.

ADD REPLYlink written 3 months ago by RamRS30k

and Kevin is my brother and I don't want to disrespect him at any cost

ADD REPLYlink written 3 months ago by anasjamshed199420

brother, what can I do if I don't find a solution from the last 10 days? I already contacted authors

ADD REPLYlink written 3 months ago by anasjamshed199420

In that case, there is nothing you can do but wait. You could send the authors a second email, but beyond that, there's nothing more you can do.

ADD REPLYlink written 3 months ago by RamRS30k
0
gravatar for Kevin Blighe
3 months ago by
Kevin Blighe67k
Republic of Ireland
Kevin Blighe67k wrote:

Hi Anas, please take a look at the output of ?topTable and you will be able to figure this out.

Kevin

ADD COMMENTlink modified 3 months ago • written 3 months ago by Kevin Blighe67k

no brother I can't find any method to select threshold P < 0.01 and fold change >2.0. plz help me

ADD REPLYlink written 3 months ago by anasjamshed199420

I am pretty sure that the parameters that you need are p.value and lfc.

If you cannot filter directly with toptable(), then just do:

subset(tT, adj.P.Val < 0.05 & abs(logFC) > 2)
ADD REPLYlink written 3 months ago by Kevin Blighe67k

after runnning this subset(tT, adj.P.Val < 0.05 & abs(logFC) > 2) no genes are found . plz help me

ADD REPLYlink written 3 months ago by anasjamshed199420
1

Then nothing is statistically significantly, and there is nothing that I can do to help in relation to this. Try to reduce these thresholds (0.05 and absolute 2)

ADD REPLYlink modified 3 months ago • written 3 months ago by Kevin Blighe67k

DEGs were selected based on the threshold P < 0.01 and fold change >2.0. This is written in the paper so I want to select like this and output should show 504 genes

ADD REPLYlink written 3 months ago by anasjamshed199420

Email the authors or re-read the paper and make sure you're classifying samples accurately.

ADD REPLYlink written 3 months ago by RamRS30k

I already did all the things that's why I am asking here AND Ram if you don't want to help others then plz not criticize other

ADD REPLYlink written 3 months ago by anasjamshed199420
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