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Old 01-25-2012, 10:09 AM   #3
Location: New York, NY

Join Date: Dec 2010
Posts: 25

Just at first glance, in your cufflinks run you specify two different parameters that will affect the FPKM calculation.
--upper-quartile-norm --max-mle-iterations 20000
I would try changing --max-mle-iterations to match cuffdiff, disabling quartile normization, and running the biological replicates through cufflinks separately to see if this difference is true. Then I would try cufflinks with the merged BAMs. Internally the same code does the quantification in both cufflinks and cuffdiff.

Also, I noticed you're looking in transcripts.gtf for cufflinks and gene_exp.diff for cuffdiff. It would be better to look in isoforms.fpkm_tracking for both cufflinks and cuffdiff, as gene_exp.diff lists quantification at the locus level while transcripts.gtf is at the isoform level.
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