CuffDiff strange output

[Pruexel]

Hi,
I hope that someone can be so gentle to help me. I'm analizing some data from RNA-Seq with TopHat and Cufflinks and I focus my attention on differently spliced genes. In particular, now I'm working on "maybe false positive", for example:

NM_001130171 Zfp207 Zfp207 chr11:80196780-80219375 day0 day3 OK 0.0000000 36.6325000 1.79769e+308 1.79769e+308 2.67658e-19 1.72370e-18 yes

this row comes from isoform_exp.diff file and the log2fold change is totally crazy because one sample is zero. So, I control the expression with expressionBarplot from cummeRbund, and the fpkm value of sample one was not zero! This function takes the information from another file, gene_exp.diff:

Zfp207 chr11:80196780-80219375 day0 day3 OK 26.4421 97.6658 1.88502 -15.6715 0 0 yes

The two files should be have the same results, but they are different and a p-value=0 is really strange.

Have someone some information about this? There is some normalization not explained in Cufflinks web site? What is the final true fpkm value?

Thanks

[muthu545]

Hi,
The gene zfp207 is differentially expressed in your comparison of day0 to day3.
Zfp207 chr11:80196780-80219375 day0 day3 OK 26.4421 97.6658 1.88502 -15.6715 0 0 yes

With respect to the isoforms of Zfp207. you are looking at the expression of only one isoform, which is
NM_001130171 Zfp207 Zfp207 chr11:80196780-80219375 day0 day3 OK 0.0000000 36.6325000 1.79769e+308 1.79769e+308 2.67658e-19 1.72370e-18 yes
This particular isoform is differentially expressed in your condition. of note, this isoform is not expressed at day0 and only shows up on day3.

Please look at the expression of other isoforms of Zfp207:
NM_001130169
NM_001130170
NM_011751
NR_045038 - may not exist in your data.

If you total the individual expression of all the isoforms (4 of them), the total RPKM should sum up to ~26 for day0 and ~97 for day3, which is the corresponding gene expression.

isoform_exp.diff summarizes the expression table and DE analysis of all the possible isoforms of genes in your study.
gene_exp.diff summaries the expression table and DE analysis of all the genes in your study.

for example: if geneA has only 1 isoform then the values in both files (iso and gene_exp.diff) will match.

In your case, you have to sum up the expression value of all the isoforms in iso_exp.diff file and the total of this will match the expression value of specific gene in gene_exp.diff.

--
Thanks

[Pruexel]

Thank you,
I find the other isoforms and returns all and thank you very much also for the other informations.