Question: Cuffdiff No isoform FPKM Tracking and differential testing
gravatar for ahdee
4.3 years ago by
United States
ahdee30 wrote:

Hi All, 

my dataset is an RNA-Seq set from illumina; workflow includes Tophat for Illumina -> Cufflinks -> CuffMerge -> Cuffdiff; my output all looks fine except I do not see isoform_exp and isoform_fpkm, strangely isoform_read_group is present though. 

I think I'm doing something wrong? Can someone help? thanks. 



rna-seq galaxy fpkm cufflinks • 2.0k views
ADD COMMENTlink modified 4.3 years ago by Jennifer Hillman Jackson25k • written 4.3 years ago by ahdee30
gravatar for Jennifer Hillman Jackson
4.3 years ago by
United States
Jennifer Hillman Jackson25k wrote:


Are you including a reference annotation file with your analysis (GTF or GFF3)? Does it include the attribute 'tss_id' (and 'p_id')? Obtain a version with these if not, and possible (exists).

Best, Jen, Galaxy team


ADD COMMENTlink written 4.3 years ago by Jennifer Hillman Jackson25k

Thanks Jen, neat point I notice that my cuffmerge output file does not contain the p_id tag, for example in one of the output row,

 class_code "="; tss_id "TSS1"; 

but does not contain the p_id tag.   Not sure if this will solve it or how to get cuffmerge to output p_id tag as well. Any thoughts on this?  Will update my results in case someone runs into the same problem. 


ADD REPLYlink written 4.3 years ago by ahdee30

To be clear, you are not using a reference annotation dataset currently? This is needed for these functions. Hopefully this will work out for you! Jen, Galaxy team


From the manual link above. Cuffmerge runs Cuffcompare (which is slightly confusing, but there are reasons for having both tools):


The ID of the coding sequence this transcript contains. This attribute is attached by Cuffcompare to the.combined.gtf records only when it is run with a reference annotation that include CDS records. Further, differential CDS analysis is only performed when all isoforms of a gene have p_id attributes, because neither Cufflinks nor Cuffcompare attempt to assign an open reading frame to transcripts.


ADD REPLYlink written 4.3 years ago by Jennifer Hillman Jackson25k
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