Question: Very large FPKM from cuffdiff that doesn't match read counts
gravatar for fcchau
3.3 years ago by
fcchau0 wrote:

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!

rna-seq • 968 views
ADD COMMENTlink modified 3.3 years ago by Petr Ponomarenko2.6k • written 3.3 years ago by fcchau0

Hello fcchau!

It appears that your post has been cross-posted to another site:

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

ADD REPLYlink written 3.3 years ago by WouterDeCoster43k

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

ADD REPLYlink written 3.3 years ago by fcchau0

How does it look in IGV ?

ADD REPLYlink written 3.3 years ago by poisonAlien2.8k

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

ADD REPLYlink written 3.3 years ago by fcchau0
gravatar for Petr Ponomarenko
3.3 years ago by
United States / Los Angeles /
Petr Ponomarenko2.6k wrote:

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

ADD COMMENTlink written 3.3 years ago by Petr Ponomarenko2.6k

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

ADD REPLYlink written 3.3 years ago by fcchau0
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