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Old 11-23-2010, 09:29 PM   #1
ngs
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Default multiple FPKM problem for single gene in gene_exp.diff after running cuffdiff

I ran tophat (1.1.0) without a mouse gtf file. Run cufflinks (0.9.1) without a mouse gtf file. Then run cuffcompare with a mouse gtf file and two gtf files generated from cufflinks for my two samples. Finally, I ran cuffdiff with compare.combined.gtf and two accepted_hits.bam files.

However, I checked one of the cuffdiff output files: gene_exp.diff. I found there is still multiple FPKM problem for many genes (see below):

XLOC_000009 Cspp1 chr1:10053629-10189988 q1 q2 OK 44.5012 58.359 0.271096 -2.93789 0.00330457 yes
XLOC_000010 Arfgef1 chr1:10053629-10189988 q1 q2 OK 10.0582 7.68137 -0.269589 4.88261 1.04688e-06 yes
XLOC_000011 Arfgef1 chr1:10053629-10189988 q1 q2 OK 40.66 31.8566 -0.244 17.6406 0 yes
XLOC_000013 Arfgef1 chr1:10053629-10189988 q1 q2 OK 2.7768 40.8059 2.68753 -144.972 0 yes
XLOC_000015 Arfgef1 chr1:10053629-10189988 q1 q2 OK 54.0345 65.0081 0.18489 -12.9339 0 yes
XLOC_000016 Arfgef1 chr1:10053629-10189988 q1 q2 OK 23.4654 43.6672 0.62107 -29.4492 0 yes
XLOC_000031 Tram2 chr1:20986216-20997026 q1 q2 OK 5.8219 2.96147 -0.67594 3.70609 0.000210487 yes
XLOC_000032 Tram2 chr1:20986216-20997026 q1 q2 OK 3.33419 14.9065 1.49757 -29.7646 0 yes
XLOC_000057 Tmem131 chr1:36849038-36996484 q1 q2 OK 37.3723 30.8444 -0.191975 5.03247 4.84195e-07 yes

Did I do something wrong? Wht do some genes only have one FPKM (one row) and some genes have multple FPKM (multple rows)?

I have another question regarding gene_exp.diff file. The first gene Cspp1 has the same coordiates (chr1:10053629-10189988) as the second gene Arfgef1. But in my mouse gtf file (from Ensembl), the coordinates for those two genes are:
Cspp1: Chromosome 1: 10,028,299-10,126,849
Arfgef1: Chromosome 1: 10,127,652-10,222,751

Those two genes are not overlapped. Why do they have the same coordinates in gene_exp.diff file?

Thank you very much!
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Old 02-10-2011, 03:50 AM   #2
reut
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Default I have the same issue of multiple lines per gene

I have this issue as well -
can anyone explain why there are multiple lines per gene?
is it valid or indicate a problem?
how can I reduce it to have 1 line per gene?

hope now someone will answer,
thanks
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Old 03-30-2011, 06:03 AM   #3
gzentner
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Default

Bump - I have been wondering this same thing myself.

I thought the gene_exp.diff was supposed to sum or average the FPKMs for each gene to give an overall expression level.
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Old 03-30-2011, 06:27 AM   #4
kmcarr
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Cuffcompare IS reporting one line per gene, but the "genes" it cares about are the XLOC models, not the ones named in column 2. The gene names from your GTF file(s) (column 2) merely report the closest annotated gene model to the models developed by cufflinks/cuffcompare.

Quote:
I ran tophat (1.1.0) without a mouse gtf file. Run cufflinks (0.9.1) without a mouse gtf file. Then run cuffcompare with a mouse gtf file and two gtf files generated from cufflinks for my two samples. Finally, I ran cuffdiff with compare.combined.gtf and two accepted_hits.bam files.
To get results which solely report previously annotated genes you should run TopHat with the "-G mm9.gtf --no-novel-junctions" and then run Cufflinks with "-G mm9.gtf"

Last edited by kmcarr; 03-30-2011 at 06:36 AM.
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Old 03-30-2011, 02:55 PM   #5
Camg
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A similar thing happened with my gene.exp.diff file where the first few genes have the correct ID's matching those in the gtf file and have different FPKM's, but the coordinates are the same (they all match that of the very first gene from .gtf).

This seems more like a glitch to me since it seems to only occur for the first few lines of the exp.diff file. Is this the same for everyone else? or did you find this phenomenon at random throughout your exp.diff file? Also, if cuffdiff were really having a problem where it considered several genes to have identical coordinates then wouldn't the FPKMs be the same as well?

I hope that makes some sense, and thanks everyone for the comments.
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