Hi All, I agree with this problem and solution. I have a lot of cufflinks, cuffcompare and cuffdiff output but I am struggling to relate what this means in terms of the real world! I have seen partek software attempt to visualise some of the data it generates which appears to be using the FMI data in the cufflinks suite but beyond that I struggle. I did have an email conversation with Cole Trapnell which eventually centred on the idea that you just have to trust the analysis and then go away and do the RT-PCR to check it all out! So for tools I think: 1. A tool that shows you the layout of known isoforms for a gene and the FMI data for each isoform. er. thats it for now from me! But I also struggle to understand what all the other outputs really mean! What does the CDS.diff output tell us? What dies the promoters.diff output tell us? I know what the cufflinks manual says but I struggle to convert this in my head to what is happening to an actual gene so if anyone has a power point example on a specific gene of what the data is saying in terms of how this relates to changes in protein production - that would be great! I'm hoping someone out there has had to lecture on this to students and they have done a powerpoint presentation and are willing to show it to the galaxy community. Another point about the analysis of cufflinks data is the subject of the Pseudo Autosomal Regions in X and Y - this will make a mess of gene expression analysis in some cases especially because tophat will assign a read to both sites and make it a multihit read (which you might then filter out) or it may double the true levels of reported expression.. Anyone had thoughts on this? Best Wishes, David. __________________________________ Dr David A. Matthews Senior Lecturer in Virology Room E49 Department of Cellular and Molecular Medicine, School of Medical Sciences University Walk, University of Bristol Bristol. BS8 1TD U.K. Tel. +44 117 3312058 Fax. +44 117 3312091 D.A.Matthews@bristol.ac.uk On 10 Mar 2011, at 15:55, Jeremy Goecks wrote:

Jagat,

Please send queries such as these to the galaxy-user mailing list (cc'd); there are many users on the list who can contribute to this discussion, and there are many additional users that will benefit from this discussion.

...

I was wondering if you can point me to a documentation or URL to guide how to perform the downstream analysis once we have cuffdiff out put.

In general, I agree that tools are needed to further process cufflinks/compare/diff outputs, but I'm not aware of any that are publicly available. Let's open this issue up for discussion and see if we can reach a consensus about tools might be useful. Everyone, please feel free to contribute ideas/tools; note that the Galaxy Tool Shed is a nice place for sharing tools you've built for Galaxy:

http://community.g2.bx.psu.edu/

...

Just like any mRNA-seq experiment to achieve following objectives:

1. Reconstruct all transcripts of a particular gene and corresponding Cuffdiff significantly expressed transcripts as called by cuffdiff. 2. What are different isoforms 3. Location of splicing

From various output files which unique ID can be matched from one file say Cuffdiff.expr (transcript/ isoform/Splicing) to other file - transcript.gtf corresponding to each sample or combined GTF file.

I've got a script that does this for the cuffdiff isoform expression testing file and a GTF file; I'll wrap it up and add it to Galaxy in the next couple weeks. It would probably be useful to have similar scripts for the other expression testing files as well. Also, it would be nice to be able to take the FPKM values generated by Cuffdiff and attach them to their respective transcripts as attributes.

Best, J.

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