We have RNA-seq data (three biological replicates in a single condition) and we want to calculate TPM with the sole purpose of ranking genes by expression level (in quantiles) .
I understand that, while TPM uses the total reads number to normalize sequencing depth and library size, for DE is taken a more sophisticated approach (TMM in edgeR or "median ratio method" in DEseq2).
I have two questions:
1) Should I normalize my counts with a "DE method" instead of relying on the total reads system?
2) Which is the best way to collapse the three replicates?
I was thinking of taking the median TPM for each gene, but there might be some smarter way.