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
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
Thanks for your reply Michael. I will check if this could help me. If not DESeq, is there any other Package you are aware of hat could be more suitable for me to use? Thank you.
I don't follow, I told you how to compute a stabilized LFC from one replicate data using DESeq2.
Hi Michael, thank you very much for your reply. I followed your suggestion have found my way to get normalised counts and calculate LFC. Thanks again :)
You can use NOISeq, which can perform differential expression analysis without replicates.
NOISeq
Refer 5.1.2 NOISeq-sim: no replicates available. Note this method will simulate replicate with normal distribution, but still better than others.