Formula to calculate FPKM

Question

[vinayjrao]

Hello,

I am working with RNA-Seq data. I have the read counts for each, and before checking for differential expression, I want to normalize it using FPKM. In theory, I understand the difference between RPKM and FPKM, but could someone please tell me a formula to calculate the same?

Thank you.

[Kevin Blighe]

An update (6th October 2018):

You should abandon RPKM / FPKM. They are not ideal where cross-sample differential expression analysis is your aim; indeed, they render samples incomparable via differential expression analysis:

Please read this: A comprehensive evaluation of normalization methods for Illumina high-throughput RNA sequencing data analysis

The Total Count and RPKM [FPKM] normalization methods, both of which are still widely in use, are ineffective and should be definitively abandoned in the context of differential analysis.

Also, by Harold Pimental: What the FPKM? A review of RNA-Seq expression units

The first thing one should remember is that without between sample normalization (a topic for a later post), NONE of these units are comparable across experiments. This is a result of RNA-Seq being a relative measurement, not an absolute one.

Answer 1

[Rahul Sharma]

Although I would use DESeq2, Cuffdiff or other published and well established methods for finding DEGs. Here is a link where they explained the formula in details: RPKM, FPKM and TPM, clearly explained

[vinayjrao]

Dear Rahul,

I have gone through the explanation provided on the link, but it still doesn't explain how you can relate read counts to fragment count. Is there any other way? As you said I could always use the tools, but I want to understand the method of calculating fpkm.

[swbarnes2]

Rahul's point is if you want to do differential expression, let the software designed to do that handle the normalization. It does a better, more sophisticated method than using RPKM. You can probably get whatever software you use to give you the results of its normalization.

[vinayjrao]

I could use a tool, that's not a problem, but I want to calculate it manually just so that I know the exact process of doing it. As I said before, I understand it theoretically, but can't calculate it myself.

Thanks.

[lovegenetics]

Dear Vinay,

once you've figured out the difference between read counts and fragments count, please share :) I need to use FPKM instead of RPKM since the experiment used was paired-end reads method.

Thank you.