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Old 04-18-2012, 04:17 AM   #5
sudders
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Location: Sheffield, UK

Join Date: Dec 2011
Posts: 32
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Did you ever find a solution to this? We run into the same problem.

Our pipeline is thus:
We map reads with tophat for each sample
Run cufflinks on each sample to generate a transcriptome assembly

the command looks something like:
Code:
 cufflinks --label tax-Pre-R5
               --num-threads 4
               --library-type fr-secondstrand
               --frag-bias-correct /ifs/mirror/genomes/bowtie/hg19.fa
               --multi-read-correct
               --upper-quartile-norm
               /ifs/projects/proj004/rnaseq4/tax-Pre-R5.accepted.bam
Run Cuffmerge and Cuffcompare to generate merged gene sets.

We also run cuff diff to test for differences.

Our cuffdiff commands look like:

Code:
 cuffdiff --output-dir abinitio.cuffdiff.dir             
                 --library-type fr-secondstrand
                 --upper-quartile-norm 
                 --frag-bias-correct /ifs/mirror/genomes/bowtie/hg19.fa
                 --multi-read-correct
                 --verbose
                 --num-threads 16
                 --labels Prostate-Pre-agg,Prostate-Post-agg,tax-Pre-agg,tax-Post-agg              
                 --FDR 0.050000
                abinitio.gtf
              Prostate-Pre-R7.accepted.bam,Prostate-Pre-R1.accepted.bam,Prostate-Pre-R4.accepted.bam,Prostate-Pre-R2.accepted.bam,Prostate-Pre-R8.accepted.bam,Prostate-Pre-R5.accepted.bam,Prostate-Pre-R3.accepted.bam,Prostate-Pre-R6.accepted.bam
             Prostate-Post-R7.accepted.bam,Prostate-Post-R8.accepted.bam,Prostate-Post-R6.accepted.bam,Prostate-Post-R3.accepted.bam,Prostate-Post-R5.accepted.bam,Prostate-Post-R2.accepted.bam,Prostate-Post-R4.accepted.bam,Prostate-Post-R1.accepted.bam   
            tax-Pre-R1.accepted.bam,tax-Pre-R3.accepted.bam,tax-Pre-R2.accepted.bam,tax-Pre-R6.accepted.bam,tax-Pre-R4.accepted.bam,tax-Pre-R5.accepted.bam
           tax-Post-R6.accepted.bam,tax-Post-R1.accepted.bam,tax-Post-R4.accepted.bam,tax-Post-R5.accepted.bam,tax-Post-R2.accepted.bam,tax-Post-R3.accepted.bam
If we compare the FPKMs coming out of cuffcompare and cuffdiff they are not even within two or three orders of magnitude of each other, with the cuffcompare FPKMs being in the millions or tens of millions, while the cuffdiff outputs being in the more sensible 0 - several hundred range.

We're using cufflinks 1.3.1.
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