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Old 01-08-2014, 08:22 AM   #1
kuintzlr
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Location: Corvallis, OR

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Default Cufflinks/Cuffdiff isoforms.fpkm_tracking output: greater FPKMs than the upper bound

I have run several different data sets through the Tuxedo pipeline, but have just noticed that the isoforms.fpkm_tracking files are all messed up in the FPKM columns. According to the Cufflinks manpage, columns 10-12 of all the tracking files are defined as follows:

10 q0_FPKM 8.01089 FPKM of the object in sample 0
11 q0_FPKM_lo 7.03583 the lower bound of the 95% confidence interval on the FPKM of the object in sample 0
12 q0_FPKM_hi 8.98595 the upper bound of the 95% confidence interval on the FPKM of the object in sample 0

However, in my files, MANY column 10 FPKM values are several orders of magnitude higher than column 12, FKPM_conf_hi, which is the upper bound. Has anyone else noticed this in their files? I cannot find any explanation for this, and like I said, all of these files, even from different datasets and different experiments, show the same phenomenon.

I am using TopHat v2.0.9, cufflinks v2.1.1.

I have attached a pdf image of a scatterplot to visualize these differences, because it seems that this error is correlated to fragment length. On the y-axis is the difference FKPM(column 10 above)-FPKM_conf_hi(column 12, upper bound). This SHOULD be negative. All points above the x axis are erroneous.
The x-axis is fragment length (Column 8 of isoforms.fpkm_tracking). You can see that this error seldom occurs at higher fragment lengths.

Please help!
Attached Files
File Type: pdf cuffdiff_figure.pdf (458.7 KB, 27 views)
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Old 01-08-2014, 06:00 PM   #2
Dario1984
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It's a known problem. The confidence intervals are calculated wrongly. The estimate is correct, though. The next version of Cufflinks will have this fixed.
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Old 01-09-2014, 10:36 AM   #3
kuintzlr
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Thank you Dario1984. Where did you find this information?
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Old 03-30-2014, 10:00 PM   #4
Dario1984
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I read it on a blog. The new version of Cufflinks is now released. You can run your analysis again.
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