Hello Everyone,

For exploratory reasons we have RNAseq data without replicates, I totally understand that it is not possible to estimate any sgnificance from this data as there are no replicates, the purpose is only for exploratory reasons. I am new to bioinformatics and would appreciate any help and sugggestions.

I would like to perform differential expression analysis to understand which genes are majorly affected (Foldchange) in the "control" versus "treatment". I have read the previous posts about this kind of analysis but without any luck.

Previous posts I have read and followed:

http://seqanswers.com/forums/showthread.php?t=31036

Deseq2 for RNAseq experiments without replicates

DESeq2, no replicate

Below is an example of the code I am running and the errors I encounter:

library(DESeq2)

countdata<-read.table("T1.txt", header=TRUE, row.names=1, check.names = F)

class(countdata)

countdata<-as.matrix(countdata)

class(countdata)

condition<-factor(c("Control,"Treatment"))

(coldata<-data.frame(row.names=colnames(countdata), condition))

coldata

dds<-DESeqDataSetFromMatrix(countData =  countdata, colData = coldata, design=~condition)

dds <- DESeq(dds)

# Error :
estimating size factors
estimating dispersions
Failure in checkForExperimentalReplicates(object, modelMatrix) : 
The design matrix has the same number of samples and coefficients to fit, so estimation of dispersion is not possible. Treating samples as replicates was deprecated in v1.20 and no longer supported since v1.22.

DESeq(dds)

# Error :
estimating size factors
estimating dispersions
Fehler in checkForExperimentalReplicates(object, modelMatrix) : 
The design matrix has the same number of samples and coefficients to fit, so estimation of dispersion is not possible. Treating samples as replicates was deprecated in v1.20 and no longer supported since v1.22.

res <- results(dds)

# Error:
Fehler in results(dds) : 
couldn't find results. you should first run DESeq()

#

Any help and pointers in this direction would really be helpful for me. If anyone could share the code to use with the latest version of DESeq2 for this kind of analysis would be great. Thank you very much in advance :)

Cheers, PD

You can calculate vst() on a dds with a design of ~1 and then subtract the two columns to form a LFC.

We don't offer any inference on design without replicates through DESeq().