Question: logCPM error in RStudio.
1
gravatar for far.zi
29 days ago by
far.zi10
USA
far.zi10 wrote:

Hi, I've beed doing RNA-Seq analysis using R. However, when I get the final edgeR_result file, the logCPM values are very high which is not true for the genes I'm studying since they are extremely low expressed. I've checked my count table many times and no mistake there. Can someone help me please to figure it out why I'm getting those high values? Thanks I also put the script I use in R in case needed:

cts <- read.csv("normCounts.txt", sep = '\t', row.names = 1, header = FALSE) 
group <- (c(1,1,1,1,2,2,2,2))
cts <- DGEList(cts, group)
#cts <- calcNormFactors(y)
design <- model.matrix(~group)
cts <- estimateDisp(cts, design)
fit <- glmQLFit(cts, design)
qlf <- glmQLFTest(fit, coef = 2)
write.table(qlf$table, file = "edgeR_result.txt")
res <- qlf$table

ggplot(res, aes(x=logCPM, y=logFC, color=PValue < 0.05)) + 
    geom_point() + geom_rug(sides = "l") + theme_minimal()

fit2 <- glmFit(cts, design)

enter image description here

rna-seq R software error • 137 views
ADD COMMENTlink modified 29 days ago by ATpoint40k • written 29 days ago by far.zi10
1

Are you feeding normalized counts into edgeR? Is this correct that you #-ed the calcNormFactors? Please add the plot you created.

ADD REPLYlink modified 29 days ago • written 29 days ago by ATpoint40k

Yes, I am feeding the normalized values.

ADD REPLYlink written 29 days ago by far.zi10

That is not correct. Please read the manual. You must start from raw counts. There are also very few points in this plot. How many in total?

ADD REPLYlink written 29 days ago by ATpoint40k

OMG, I thought I need the norm counts. The total is 478.

ADD REPLYlink written 29 days ago by far.zi10

That is a very low gene number, so this is no normal RNA-seq, right? Yes, raw counts, I suggest you go through the edgeR manual first before continuing.

ADD REPLYlink modified 29 days ago • written 29 days ago by ATpoint40k

Those are "Mirtron" reads. I'll also check the manual again. Thank you :)

ADD REPLYlink written 29 days ago by far.zi10
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