Question: Differential gene expression analysis problem in EDGE R?
0
gravatar for harshraje19
4 weeks ago by
harshraje1920
harshraje1920 wrote:

Dear all,

I have performed the small RNA sequencing and I found 4000 known miRNAs and 35 novel miRNAs.

But when I am performing the EDGE R test in R-studio it is working for known miRNAs but not working for novel miRNAs, is it because of their numbers are less? and also error is null hypothesis

Can anyone help me reading this.

Thanks and regards Raj

software error rna-seq R gene • 165 views
ADD COMMENTlink modified 4 weeks ago • written 4 weeks ago by harshraje1920

Please add relevant code. Anecdotal descriptions are difficult to follow. Try to better explain what exactly the problem is. What do yo mean by and also error is null hypothesis? Please edit your questions to address these questions.

ADD REPLYlink written 4 weeks ago by ATpoint31k

How did you identify these '4000 known and 35 novel' miRNAs and why are you performing differential expression analysis separately ?

ADD REPLYlink modified 4 weeks ago • written 4 weeks ago by Arup Ghosh2.3k

Actually I am performing the DEG in combine, but it is giving following error

 library(edgeR)
 Counts<- read.table("m1.txt",sep = "\t",stringsAsFactors = F,row.names = 1)
 colnames(Counts)<-paste("P",c(81,87,92,97,103,83),sep = "")
 deg_list <- DGEList(counts=Counts, genes=rownames(Counts))
 ordered <- order(rowSums(deg_list$counts), decreasing=TRUE)
 ##udating library size
 deg_list$samples$lib.size <- colSums(deg_list$counts)
 ##udating library size
 deg_list$samples$lib.size <- colSums(deg_list$counts)
 keep <- filterByExpr(deg_list)
 deg_list <- deg_list[keep, , keep.lib.sizes=FALSE]
 deg_list=calcNormFactors(deg_list,method="TMM")
 # DESIGN MATRIX
 group=factor(c(rep(1,3),rep(2,3),rep(3,3),rep(4,3)))
 designMatrix<- model.matrix(~group, data=factor(rownames(deg_list$samples)))
 deg_list<- estimateDisp(deg_list, designMatrix, robust=TRUE)
  Error in glmFit.default(sely, design, offset = seloffset, dispersion = 0.05,  : 
  nrow(design) disagrees with ncol(y)
ADD REPLYlink modified 29 days ago by ATpoint31k • written 4 weeks ago by harshraje1920

Please show colnames(Counts). Your design matrix (so the number of indicated replicates/the groups) do not agree with the dimensions of the count table, meaning you have more or fewer samples than indicated in the design.

ADD REPLYlink written 29 days ago by ATpoint31k

This is my colnames(Counts)

colnames(Counts)<-paste("P",c(81,87,92,97,103,83),sep = "")

ADD REPLYlink modified 29 days ago • written 29 days ago by harshraje1920
2
gravatar for ATpoint
29 days ago by
ATpoint31k
Germany
ATpoint31k wrote:

So 6 samples, but here group=factor(c(rep(1,3),rep(2,3),rep(3,3),rep(4,3))) you defined 3*4 groups, why is that?

ADD COMMENTlink written 29 days ago by ATpoint31k

Oh then it was my mistake then, then should be

group=factor(c(rep(1,3),rep(2,3)))

right??

ADD REPLYlink written 29 days ago by harshraje1920

WOW. It is working after changing the group factor to

group=factor(c(rep(1,3),rep(2,3)))

Thank you very much

Raj

ADD REPLYlink written 29 days ago by harshraje1920

If an answer was helpful, you should upvote it; if the answer resolved your question, you should mark it as accepted. You can accept more than one if they work.
Upvote|Bookmark|Accept

ADD REPLYlink written 29 days ago by Arup Ghosh2.3k
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