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Question: Cuffdiff No isoform FPKM Tracking and differential testing
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gravatar for ahdee
4.3 years ago by
ahdee • 30
United States
ahdee • 30 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. 

 

Ahdee
rna-seq galaxy fpkm cufflinks • 2.0k views
ADD COMMENT • link •
modified 4.3 years ago by Jennifer Hillman Jackson ♦ 25k • written 4.3 years ago by ahdee • 30
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gravatar for Jennifer Hillman Jackson
4.3 years ago by
Jennifer Hillman Jackson ♦ 25k
United States
Jennifer Hillman Jackson ♦ 25k wrote:

Hello,

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).
http://cufflinks.cbcb.umd.edu/manual.html#cuffdiff_input

Best, Jen, Galaxy team

 
ADD COMMENT • link written 4.3 years ago by Jennifer Hillman Jackson ♦ 25k

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. 

'ahdee

ADD REPLY • link written 4.3 years ago by ahdee • 30

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):

p_id

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 REPLY • link written 4.3 years ago by Jennifer Hillman Jackson ♦ 25k
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