Question: quantifying differential gene expression across three groups using cuffdiff
0
gravatar for drew.halfpenny
2.9 years ago by
United States
drew.halfpenny0 wrote:

Hello,

I am trying to identify differentially expressed genes across three groups.  The groups consist of RNA-seq data from a cell line that was treated with protein A, protein B, or untreated.  Each group contains three samples.  I mapped the samples individually to reference genome hg19 using tophat.  I then attempted to use cuffdiff to identify differentially expressed genes using the igenomes hg19 genes file as a reference notation and 3 concatenated files containing the mapped BAM files for the 3 samples per group as 3 distinct conditions (treated A, treated B, untreated).  I did not run the cufflinks assembler or cuffmerge on the data because I am not concerned with novel genes, just known and differentially expressed genes.  The two proteins are known to cause cancerous-like mutations and this should be evident from differentially expressed genes.  The cuffdiff output of files containing information about differentially expressed genes only compared 2 of the three groups.  The cuffdiff output of files containing information about FPKM values compared all three of the groups.  Is it necessary to run cuffdiff 3 separate times for this analysis?  The resultant data from 3 separate runs of cuffdiff is much more difficult to analyze.  Any suggestions would be greatly appreciated.

Drew

rna-seq galaxy cuffdiff • 1.3k views
ADD COMMENTlink modified 2.9 years ago by Jennifer Hillman Jackson25k • written 2.9 years ago by drew.halfpenny0
0
gravatar for Jennifer Hillman Jackson
2.9 years ago by
United States
Jennifer Hillman Jackson25k wrote:

Hello,

Cuffdiff compares two conditions at time as available on the public Galaxy server http://usegalaxy.org

Just as a note, it is not clear if you concatenated three Tophat results per condition (merging 3 samples). If so, it is better to run these as distinct samples without merging. If I misunderstood, please ignore this part!

Thanks, Jen, Galaxy team

ADD COMMENTlink written 2.9 years ago by Jennifer Hillman Jackson25k
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