Question: Formula to calculate FPKM
2
gravatar for vinayjrao
2.4 years ago by
vinayjrao170
inStem, India
vinayjrao170 wrote:

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.

rna-seq fpkm • 4.3k views
ADD COMMENTlink modified 2.4 years ago by Rahul Sharma600 • written 2.4 years ago by vinayjrao170

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.

ADD REPLYlink modified 22 months ago • written 24 months ago by Kevin Blighe63k
1
gravatar for Rahul Sharma
2.4 years ago by
Rahul Sharma600
Germany
Rahul Sharma600 wrote:

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

ADD COMMENTlink written 2.4 years ago by Rahul Sharma600

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.

ADD REPLYlink written 2.4 years ago by vinayjrao170

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.

ADD REPLYlink written 2.4 years ago by swbarnes28.1k

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.

ADD REPLYlink written 2.4 years ago by vinayjrao170

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.

Many thanks,

Reem Alhamidi

ADD REPLYlink written 12 months ago by alhamidi.reem20
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