how to calculate fpkm?

Question

[vinayjrao]

Hi,

I am working with paired-end reads and I have to now calculate FPKM. My question is, how to calculate FPKM. wc -l mcf7_1.fastq gave me 26723524 reads and wc -l mcf7_2.fastq gave me 26723524 reads as well (I divided the wc -l output by 4), but for FPKM, do I need to take only 26723524 fragments or 53447048?

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

[venu]

It is not possible to calculate FPKM from raw fastq files. You need to align the data to a reference genome. Then you'll get a BAM file. There are many tools out there to calculate FPKM using BAM files.

Here is your guide on how to calculate FPKM.

[vinayjrao]

Thanks Venu, but that part I know. I need to know what value to take to calculate the number of fragments. I have also seen the video in the link provided, but it only explains RPKM and TPM.

Answer 2

[lakhujanivijay]

There is a very good explanation on youtube

[vinayjrao]

Dear Vijay, the link shared by you and Venu are the same. Could you please help me locate the number of fragments and fragments mapped?

Thank you.