RPKM, FPKM, etc.

[rgregor]

Hi all,

can anybody provide a clear definition of:

* difference of RPMK vs FPKM
* describe the algorithm of how cufflinks computes FPKM

thank you,
Gregor

[Trudy]

Look this:

http://cufflinks.cbcb.umd.edu/howitworks.html

"In RNA-Seq experiments, cDNA fragments are sequenced and mapped back to genes and ideally, individual transcripts. Properly normalized, the RNA-Seq fragment counts can be used as a measure of relative abundance of transcripts, and Cufflinks measures transcript abundances in Fragments Per Kilobase of exon per Million fragments mapped (FPKM), which is analagous to single-read "RPKM", originally proposed in:

* Ali Mortazavi, Brian A Williams, Kenneth McCue, Lorian Schaeffer and Barbara Wold Mapping and quantifying mammalian transcriptomes by RNA-Seq Nature Methods, volume 5, 621 - 628 (2008)

You could read also Supplementary Material where there is the RPKM formula


In paired-end RNA-Seq experiments, fragments are sequenced from both ends, providing two reads for each fragment (FPKM)
so
RPKM=single read (single end)
FPKM=fragment are 2 reads (Paired-end)

it's clear?

[ikarus97]

So, given the same alignment of paired-end data, RPKM values are twice of FPKM value?

[Simon Anders]

So, given the same alignment of paired-end data, RPKM values are twice of FPKM value?

No, not at all.

Please read the cufflinks paper for a complete explanation:

Trapnell et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation
Nature Biotechnology doi:10.1038/nbt.1621

[ikarus97]

Got it. I stupidly thought FPKM as 'Fragments Per Kilobase of exon per Million "Reads" mapped.'

Thanks for quick answer.