Question: EdgeR mistake- dispersion
0
gravatar for trumbia
4 months ago by
trumbia0
trumbia0 wrote:

I am trying to conduct edgeR and Iam facing with 'Warning message: In estimateDisp.default(y = y$counts, design = design, group = group, : No residual df: setting dispersion to NA' mistake and my code is

files <- c("ABCDEFGHIJKLMNOPRSTU1.txt", "ABCDEFGHIJKLMNOPRSTU2.txt", 
           "ABCDEFGHIJKLMNOPRSTU3.txt", "ABCDEFGHIJKLMNOPRSTU4.txt" ) 
read.delim(files[1])
library(limma) 
library(edgeR) 
x <- readDGE(files, columns=c(1,3)) 
class(x)
dim(x)
samplenames <- substring(colnames(x), 12, nchar(colnames(x))) 
samplenames
colnames(x) <- samplenames
group <- as.factor(c("Blue", "Blue1", "Normal", "Normal1")) 
x$samples$group <- group 
lane <- as.factor(rep(c("LB1","Ln1"), c(2,2))) 
x$samples$lane <- lane 
design <- model.matrix(~0+group)
colnames(design) <- levels(group)
design
keep <- filterByExpr(x, design)
table(keep)
x <- calcNormFactors(x)
x$samples
AveLogCPM <- aveLogCPM(x)
hist(AveLogCPM)
x <- calcNormFactors(x)
x$samples
pch <- c(0,1,2,15,16,17)
colors <- rep(c("darkgreen", "red", "blue"), 2)
plotMDS(y, col=colors[group], pch=pch[group])
legend("topleft", legend=levels(group), pch=pch, col=colors, ncol=2)
plotMD(y, column=1)
abline(h=0, col="red", lty=2, lwd=2)
x
x <- estimateDisp(x, design, robust=TRUE)# I got the mistake here
fit <- glmQLFit(x, design, robust=TRUE)
B.LvsP <- makeContrasts(Blue-Normal, levels=design)
res <- glmQLFTest(fit, contrast=B.LvsP)
edger • 156 views
ADD COMMENTlink modified 4 months ago by Gordon Smyth39k • written 4 months ago by trumbia0
Answer: EdgeR mistake- dispersion
0
gravatar for Gordon Smyth
4 months ago by
Gordon Smyth39k
Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia
Gordon Smyth39k wrote:

You are making a mistake in the way that you define factors. Rather than my simply correcting your code, I will try to help you understand the issue by asking you question:

Do you have two groups or four groups? If there are only two groups, why you do define four different group names: Blue, Blue1, Normal and Normal1?

When you print out design, did you see two columns or four columns? (I know the answer to this question, I just want you to think about it.)

If you look at any edgeR or limma examples, you will see see the right way to do it.

ADD COMMENTlink modified 4 months ago • written 4 months ago by Gordon Smyth39k

I have 2 different replica for group example also I wanted to see if there is a difference between those group. That s why I consider them Blue1 and blue.

             group lib.size norm.factors lane
LMNOPRSTU1    Blue   783363            1  LB1
LMNOPRSTU2   Blue1   797764            1  LB1
LMNOPRSTU3  Normal  1511586            1  Ln1
LMNOPRSTU4 Normal1   709481            1  Ln1

This is desing matrix with 4 colums

ADD REPLYlink modified 4 months ago by Gordon Smyth39k • written 4 months ago by trumbia0

No, that isn't the design matrix. I'm going to make the same suggestion that Mike Love gave you, which is that you would be well advised to consult a bioinformatician or statistician at your own university or institution for help.

ADD REPLYlink modified 4 months ago • written 4 months ago by Gordon Smyth39k

I am not a university student at all. I was curious about this data and wanted to analyse it. If I had option to ask someone I knew, I d do this . Could you explain me how should I correct this? Thanks

ADD REPLYlink modified 4 months ago • written 4 months ago by trumbia0
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