Hello,
I'm having some problems to understand the Cufflinks FPKMs... I use Cufflinks to obtain the FPKMs froma a ".sam" file obtained from the denovo assembly permormed wit MIRA3 of a transcriptome. Cufflinks give me us an output the files "Isoforms.fpkm_trasckin" and "genes.fpkm_tracking", in both files the FPKMs values are the same. However, I tried to apply the formula FPKM=10^9*numreads_of_the_fragment/(total_assembled_reads * length_of_the_fragment)) and the values are ttally different, with non apparent correlation...
I give you an example (Num. reads assembled: 61725)
contig num.reads length FPKM(formula applied) FPKM(cufflinks)
c1 7 487 232.8670171315 402.01
c2 6 446 217.9492069373 399.86
c3 5 486 166.6758338375 287.15
c4 6 489 198.7839392516 342154.00
c5 7 712 159.2784232346 223638.00
Can somebody explain me these differences? What formula Cufflinks use? Thanks!
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Bio_ysl •
2 answers
Did you read the cufflinks paper? It's a lot more complicated than that... Not sure it's really possible to put it into a text box, even if I did claim to understand it. Supplementary methods section 3, lots of math.
Two things that may be responsible for the discrepancy:
- Handling of multimapped reads. I believe that Cufflinks evenly divides a read between all places that it maps. For example, a read that is mapped to 4 locations is counted as .25 reads at each individual locus.
- If any of your transcripts overlap (i.e. one gene has multiple transcripts), then Cufflinks does some sort of deconvolution to best determine which transcript a read belongs to.
Your read counting program probably addresses these 2 cases differently than Cufflinks.
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I think that you meant 10^6 instead of 10^9. Also, check the normalization method you used with cuffdiff. I f you used the default you actually applied a further normalization (which is better than FPKM). You can read the manual page: http://cufflinks.cbcb.umd.edu/manual.html#library_norm_meth
I think 10^9 is okay, isn't it 1000*1000000?
By the way, there should be SOME correlation with the cufflinks RPKM and the one you calculate yourself... If not, something's wrong.
Yes, 10^9 should be correct... If there is any correlation I couldn't see it, and this is what creates me so many doubts...
Ooops. Yes, that's correct...