Hi Dr. Linney,

The values for FPKM are relative to each experiment: the set of transcripts/genes assembled and evaluated, the normalization methods used. For example, when different samples are input, a different set of isoforms per gene can result from the assembly.

The manual, FAQ, and 'how it works' help to explain in more detail, but in short, this is expected with many of the ways that these tools are used. Any assembly or normalization method, that impacts the FPKM calculation, is a factor. http://cufflinks.cbcb.umd.edu

Running all the samples together is one option, to have values on the same relative scale. Or, if you wanted to skip discovery, and you have a reference annotation file to use as the "truth" assembly (best), this simplified protocol might be a fit for you:
http://cufflinks.cbcb.umd.edu/tutorial.html#differential

Hopefully this helps! Others are welcome to post comments/more suggestions,

Jen
Galaxy team

On 2/15/14 8:26 AM, Elwood Linney wrote:

Hello,

I am sure this has come up before and maybe I missed the answer.

If in using cuffdiff I run a sample 1(3 repeats) vs a sample 2 and then run the same sample 1(3 repeats) against a sample 3 or a sample 4, I get different value for fpkm from sample 1 each time.

Is there something going wrong here or is there something in the program that causes this difference?

I have seen this with online Galaxy and with the instance I have on my Mac Pro

el linney

Duke University
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