Greeting, i wanted to make a diffrential gene expression analysis using Limma package,

sample_info <- read.csv("matrix.csv" , header= TRUE, row.names = 1)
Group <- factor(sample_info$group)
design <- model.matrix(~Group)

But i got this error message:

Error in `contrasts<-`(`*tmp*`, value = contr.funs[1 + isOF[nn]]) : 
  contrasts can be applied only to factors with 2 or more levels

Just to mention, in my ''sample_info'', the only factor thar i have is ''Group'' which contains ''Low_Grade'' and ''High_Grade"

Thank you in advance.

This is actually an error message from base R rather than from limma. The error says that Group contains 0 or 1 levels, so your assumption that it contains 'Low_Grade'' and ''High_Grade" cannot be true.

Please type

levels(Group)

or

table(Group)

to see what the levels of Group actually are.

This is just a wild guess, but one possibility is that you have simply forgotten to capitalize Group, i.e., you used

Group <- factor(sample_info$group)

when

Group <- factor(sample_info$Group)

would have been correct. You can type names(sample_info) to confirm which name is correct.

In general, it would much quicker to you to trouble-shoot simple errors like this at your end rather than asking for help on a public forum. If Group gives an error when input to a standard function like model.matrix, then have a look at what Group contains. The reason for the error should become quickly apparent.