gravatar for Kai_Qi

4 hours ago by

Chicago, IL

I am using DEseq2 to do differential analysis from RNA-seq data across 4 developmental stages, each stages have 2 replicates. I can following through the manual to do comparisons between each two stages. Now I want to do the comparison across 4 stages in a time series manner so that I can know the genes that have the expression level either all the way up or down or no obvious trend.
I searched the manual and questions posted: in the manual, it usually has two conditions eg design= ~strain+minute or different treatments at different time points, mine have only 1 condition:
I directly generated the deseqDataset in this way:

ddsMat <- DESeqDataSetFromMatrix(countData = countdata,
                                 colData = coldata,
                                 design = ~ time)
dds <- ddsMat
#explore sample relationships, this is not the step for differential analysis
dds <- dds[rowSums(counts(dds)) > 1,] # remove the sample that reads from all group are 0

Then I typed:

ddsTC <- DESeq(dds, test = "LRT", reduced = ~time)

But I got the error:

> estimating size factors estimating dispersions gene-wise dispersion
> estimates mean-dispersion relationship final dispersion estimates
> fitting model and testing Error in nbinomLRT(object, full = full,
> reduced = reduced, quiet = quiet,  :    less than one degree of
> freedom, perhaps full and reduced models are not in the correct order

I searched around and in most situations the answer is advising to consult the statistician without solution to this problem. I am wondering is there a way to make it work and get a chance to see the results?



4 hours ago


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