Difference in total normalized counts for replicates - DeSeq2

Question

[urmi208]

Hello,

I have extracted the normalised counts for my RNAseq samples using Deseq2 with standard recommended command

dds <- estimateSizeFactors(dds)
counts(dds, normalized=TRUE)

However, when I sum up the total number of normalised counts per sample, I am seeing a differences in the latter for replicates. For example, sample A1 total = 40 million, whereas A2 total = 50 million. Is this normal? Why are they so different?

Thanks for any help you can provide.

[Devon Ryan]

FYI, that won't be identical, just in the same vague range.

Answer 1

[WouterDeCoster]

I don't know what variable you are using, but you didn't assign the normalised counts to anything. I guess you want something like:

normcounts <- counts(dds, normalized=TRUE)

Answer 2

[urmi208]

Yes, that's correct. Sorry I missed that.