Hello,

Can you please help me solve this issue?

I am flummoxed.

I have ran DESeq2 and have my count matrix and now want to rlogTransform to visualise the data on a PCA.

However I get the following error after simply trying to r transform the deseqReadsCount.

Code below, any help greatly appreciated!

ddsMF = DESeqDataSetFromMatrix(countData = counts, colData = colData, design = ~ type + condition)

## the formula of "design" defines the interested factor is "condition"
ddsMF$condition = relevel(ddsMF$condition, ref="72E8")
size_factors = estimateSizeFactorsForMatrix(normFactor.E8.72.E6.72)
sizeFactors(ddsMF) = size_factors
ddsMF <- DESeq(ddsMF,betaPrior = TRUE) ###added betPrior = true as suggested by Daniel gaffney
deseqResultMF <- results(ddsMF)

res <- results( ddsMF )
plotMA( res, ylim = c(-3, 3) )

ddsMF
write.csv( as.data.frame(res), file="Nottingham Combined_E8 72 E6 72_DESeq2results.csv" )

deseqNormFactor = sizeFactors(ddsMF)   ### get DESeq2 normalize factors
deseqReadsCount = counts(ddsMF, normalized=T)  ### get DESeq2 normalized reads counts

rld <- rlogTransformation(deseqReadsCount, blind=TRUE)

Error in (function (classes, fdef, mtable)  : 
  unable to find an inherited method for function ‘sizeFactors’ for signature ‘"matrix"’

You can check over the workflow:

http://www.bioconductor.org/help/workflows/rnaseqGene/#the-rlog-and-variance-stabilizing-transformations

Note that, in the workflow, we call rlog() on a 'dds' object, like so:

rld <- rlog(dds)

Above you are calling the function on a matrix. rlog() will take care of normalization of the counts for you.