Cuffdiff Input Files?

[ccard28]

Hello,
I was wondering if I could get some clarification on cuffdiff input files. I am just trying to compare 2 samples using cuffdiff so it is really straightforward. I only really have experience using tophat and cufflinks with our samples.

If I understand cuffdiff correctly I can just input the accepted hits tophat files for each sample as the aligned RNA-Seq reads. This is that straightforward for these inputs correct?

When it comes to the transcripts input file I am a little confused. I know there is a lot of confusion on using either cuffmerge or cuffcompare files from the cufflinks runs but can I not just use the UCSC all_mrna(genome) gtf file that I downloaded from UCSC's table browser for the transcripts input file? This is the file I use as my transcript reference when running cufflinks, couldn't you just go from tophat right to cuffdiff using this UCSC all_mrna gtf file and not even have to run cufflinks? I have done analysis using this method and skipping the cuffcompare/cuffmerge scenario but I want to make sure this is actually an acceptable input file for the transcripts gtf file. Galaxy for example says this input file must be "A transcript GFF3 or GTF file produced by cufflinks, cuffcompare, or other source.". Is the UCSC gtf file for all_mrna an acceptable "other source"?

Thank you for any help clarifying cuffdiff input files for me. I need to make sure I do my analysis correctly and though this makes sense to me I am not sure it is an acceptable method.

Thank You,
Chris

[DonDolowy]

I recommend downloading the reference genome from iGenome (see Tophat´s site). And yes, you can go directly from Tophat to Cuffdiff if you just want to check for differentially expressed genes without any de novo discovery. It is all very nicely described on the Cufflink site in the tutorial.

http://cufflinks.cbcb.umd.edu/tutorial.html