Re: Downstream Analysis Of Cuffdiff Out Put

Question: Re: Downstream Analysis Of Cuffdiff Out Put

7.7 years ago by

Jeremy Goecks • 2.2k 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. 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/ 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.

rna-seq cufflinks • 1.3k views

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modified 7.6 years ago • written 7.7 years ago by Jeremy Goecks • 2.2k

7.7 years ago by

David Matthews • 630

United Kingdom

David Matthews • 630 wrote:

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

ADD COMMENT • link written 7.7 years ago by David Matthews • 630

7.7 years ago by

David Matthews • 630

United Kingdom

David Matthews • 630 wrote:

Hi, Just wondering when the tophat portion of Galaxy will be updated? Its currently version 1.1.1 and there is now a version 1.2.0 (in fact I think there have been 4 updates). Cheers David

ADD COMMENT • link written 7.7 years ago by David Matthews • 630

Hi David, Will be updated to 1.2.0 when main is next updated (soon). Once implemented, feedback about how the update functions would be welcomed, Best, Jen -- Jennifer Jackson http://usegalaxy.org http://galaxyproject.org

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7.6 years ago by

Jeremy Goecks • 2.2k

Jeremy Goecks • 2.2k wrote:

Hello all, I've added a tool called 'Filter GTF file by attribute values list' to the galaxy-central code repository. This tool is available on our test server ( http://test.g2.bx.psu.edu/ ) at Filter and Sort --> GFF --> Filter GTF data by attribute values list and will be available on our main server in the next few weeks. As expected, this tool filters a GTF file based on a list of attribute values--or filters using a tabular file where attribute values are first column, as is the case for Cuffdiff output files. Potential attributes that can be filtered on include transcript_id, gene_id, tss_id, and p_id; conveniently, these are the IDs that Cuffdiff uses in its output files. Here's an example workflow: (1) Run Cufflinks/compare/diff (2) Filter Cufflinks isoform differential expression file for transcripts that are differentially expressed; in other words, filter for c12=='yes' (2) Use 'Filter GTF data by attribute values list' to filter Cuffcompare combined transcripts using the filtered file from step (2) as the attribute values list and, voila, you have a GTF file of the differentially expressed transcripts that you can view in your favorite genome browser. Hope this helps; feedback is always welcome. Best, J.

ADD COMMENT • link written 7.6 years ago by Jeremy Goecks • 2.2k

7.6 years ago by

Jeremy Goecks • 2.2k

Jeremy Goecks • 2.2k wrote:

Vasu, Please reply to the mailing list as emails to individual Galaxy developers often get lost, and there are others on the list that might be able to help you or benefit from this discussion. Now, to your question: you're using the wrong GFF filtering tool, which is an easy mistake to make as there are many of them. You want to use Filter and Sort --> GFF --> Filter GTF data by attribute values list. Using this tool, I was able to filter dataset 11--a GTF file produced by Cuffcompare--using a Cuffdiff isoform expression file (dataset 10) on transcript_id. I've shared the modified history with you. Best, J.

ADD COMMENT • link written 7.6 years ago by Jeremy Goecks • 2.2k

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