I have a question about DESeq2 count normalization in the context of 3'-end RNA sequencing. I understand that the median of rations normalization used by DESeq2 is not suitable for within-sample comparison of gene expression, notably because it does not account for differences in gene length.
However, when using 3'-end RNAseq, we end up with counts that reflect the molecular abundance of transcripts (1 molecule = 1 read), which is a big difference with traditional RNAseq in which (1 molecule = n reads).
In this context, can we do within sample comparison of gene expression of DESeq2-normalized counts, or are there factors other than gene length that are still unaccounted for and that would prevent such comparisons?
Thank you in advance,
All the best, Theo