Differential gene expression analysis problem in EDGE R?

Question

[harshraje19]

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

[ATpoint]

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.

[Arup Ghosh]

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

[harshraje19]

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)

[ATpoint]

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.

[harshraje19]

This is my colnames(Counts)

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

Answer 1

[ATpoint]

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?

[harshraje19]

Oh then it was my mistake then, then should be

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

right??

[harshraje19]

WOW. It is working after changing the group factor to

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

Thank you very much

Raj

[Arup Ghosh]

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