How to exercise RPKM cut-off

Question

[Neu]

Hi, I have 26 technical replicates. I have calculated RPKM values for each gene. Next, how to put the cut-off. Should I plot density plot of the log-trans RPKM value of all the genes and chose the peak as the cut off ? What is the standard procedure?

Thank you

[Asaf]

The standard procedure is not to use RPKM at all. There are much better ways to normalize read counts.

Answer 1

[Matteo Schiavinato]

So you have calculated RPKM for each gene for each sample. First, I would suggest you to use better normalization techniques, such as TPM ( http://www.rna-seqblog.com/rpkm-fpkm-and-tpm-clearly-explained/ ).

To set cutoffs, it always depends on your data. Making a density plot of the TPMs of each sample and comparing them will help you in understanding where the mean is, and therefore how much you lose with certain cutoffs. F.e. with FPKM a common practice was to select only FPKMs >= 1, with TPM as far as I know 1 works but requires a plot beforehand because it's a merely arbitrary number.

[Neu]

Thank you so much. I plotted density of log2 values of TPM for each sample and selected 0.06 as the cut-off.