Hi all,
I have done differential expression analysis with edgeR and now I'm trying to extract FPKM value from this tool, but it gave me an error all the time. Could you please take a look at the code and help me to solve the problem.
targets <- read.delim("targets.txt", stringsAsFactors=FALSE)
targets3
Label Treatment line time
1 CS control sensitive 0
2 DS6 drug sensitive 6
3 CT control tolerant 0
4 DT6 drug tolerant 6
5 DT24 drug tolerant 24
expressioncount <- read.delim("rawcount.txt", row.names=1)
colnames(expressioncount) <- substring(colnames(expressioncount),1,6)
dim(expressioncount)
[1] 78970 6
> head(expressioncount)
length CS DS6 CT DT6 DT24
c51581_g1_i1 462 0 2 1 1 0
c19380_g1_i1 439 3 0 2 0 2
c4832_g1_i1 665 0 2 0 7 23
c25894_g1_i1 402 0 1 1 1 3
c37752_g4_i1 736 2 4 2 3 2
c557_g1_i1 434 7 0 4 0 0
y <- DGEList(expressioncount[,2:6])
keep <- rowSums(cpm(y) > 1) >= 1
y <- calcNormFactors(y)
y$samples
group lib.size norm.factors
CS 1 20146232 1.1727230
DS6 1 19308652 0.9188417
CT 1 19792940 1.2271948
DT6 1 19051509 0.9463686
DT12 1 17752012 0.7990795
RPKM <- rpkm(y)
**Error in rpkm.DGEList(y) : Gene lengths not found**
Please kindly share me your helpful comments and suggestions.
Thank you in advance
2 answers
help(rpkm), which will tell you that you need to input lengths for each of the genes.
The lengths aren't in y, so rpkm can't see them. You want rpkm(y, gene.length=expressioncount$length).
If you have count table with length column in the end (tab-separated), try this script.
Note:
1) Header should start with #
2) First column gene name followed by counts in other columns ans the last colum with length.
https://github.com/santhilalsubhash/rpkm_rnaseq_count/blob/master/README.md
Sorry that I could not able to help you using edgeR.
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