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Old 02-09-2017, 10:02 AM   #1
fcchau
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Location: Boston

Join Date: Feb 2017
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Default Very large FPKM from cuffdiff that doesn't match read counts

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 4.2. 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!
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Old 02-16-2017, 08:37 AM   #2
fanli
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Location: California

Join Date: Jul 2014
Posts: 198
Default

I think there is a decent amount of evidence out there that cuffdiff is less than optimal for gene-level DE analysis.

See:
https://genomebiology.biomedcentral....-2013-14-9-r95
http://journals.plos.org/plosone/art...l.pone.0103207
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4914128/

If you are only doing DGE, I'd recommend edgeR or DESeq2
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