Very large FPKM from cuffdiff that doesn't match read counts

Question

[fcchau]

Hi Everyone,

I am relatively new to RNAseq, and hope can get some help from more experienced people here.

So I used cuffdiff to look at differential gene expression. I have 2 conditions and 3 replicates for each condition. When I look at the genes_read_group_tracking file, I found that for some genes, one of the replicates has very large FPKM values that doesn't match raw frags count.

For example this is what I see:

tracking_id condition replicate raw_frags internal_scaled_frags external_scaled_frags FPKM effective_length status

• XLOC_009487 WT 0 4 4.21548 4.21548 1150.43 - OK
• XLOC_009487 WT 1 5 5.39083 5.39083 1.14629 - OK
• XLOC_009487 WT 2 8 7.56804 7.56804 1.60234 - OK
• XLOC_009487 OE 1 2 2.29124 2.29124 0.476229 - OK
• XLOC_009487 OE 0 5 4.84785 4.84785 0.995213 - OK
• XLOC_009487 OE 2 5 4.07315 4.07315 0.77989 - OK

You can see that the FPKM for WT 0 is 1150 where as the raw frags is only 4. The other samples are fine. I observed this in multiple genes, and they don't always happen to the same sample. I also check the FPKMs from cufflinks, and they look normal. So seems that it's cuffdiff's problem.

Does anyone know why this happen and how to solve it? Appreciate the help!

[WouterDeCoster]

Hello fcchau!

It appears that your post has been cross-posted to another site: http://seqanswers.com/forums/showthread.php?t=74140

This is typically not recommended as it runs the risk of annoying people in both communities.

[fcchau]

Sorry about that, I'll keep that in mind in the future

[poisonAlien]

How does it look in IGV ?

[fcchau]

There are very few reads when I look in IGV, so the raw frags of 4 seems to make sense. the FPKM can't be more than 1000

Answer 1

[Petr Ponomarenko]

Do you get very high FPKM values for that experiment using other versions of cufflinks/cuffdiff?

[fcchau]

I was using cufflinks 2.2.1 in command line. Later I tried to use cufflinks in Galaxy, settings are slightly different

I get inflated FPKM in both cases, though the magnitude is different, and some of the genes are different