Get Wig File After Tophat

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Question: Get Wig File After Tophat

7.8 years ago by

Ying Zhang • 70

Ying Zhang • 70 wrote:

Hi: I am using tophat in galaxy to analyze my paired-end RNA-seq data and find out that after the tophat analysis, we can not get the wig file from it anymore which is used to be able to. Do you have any idea of how to still be able to get the wig file after tophat analysis? Thanks a lot! Best Ying Zhang, M.D., Ph.D. Postdoctoral Associate Department of Genetics, Yale University School of Medicine 300 Cedar Street,S320 New Haven, CT 06519 Tel: (203)737-2616 Fax: (203)737-2286

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modified 7.8 years ago by Jeremy Goecks • 2.2k • written 7.8 years ago by Ying Zhang • 70

7.8 years ago by

Hiram Clawson • 260

Hiram Clawson • 260 wrote:

FYI: samtools + bam file + a bit of perl == to wiggle conversion: https://lists.soe.ucsc.edu/pipermail/genome/2010-December/024318.html To: galaxy-user@bx.psu.edu Subject: [galaxy-user] get wig file after tophat Hi: I am using tophat in galaxy to analyze my paired-end RNA-seq data and find out that after the tophat analysis, we can not get the wig file from it anymore which is used to be able to. Do you have any idea of how to still be able to get the wig file after tophat analysis? Thanks a lot! Best Ying Zhang, M.D., Ph.D. Postdoctoral Associate Department of Genetics, Yale University School of Medicine 300 Cedar Street,S320 New Haven, CT 06519 Tel: (203)737-2616 Fax: (203)737-2286 _______________________________________________ The Galaxy User list should be used for the discussion of Galaxy analysis and other features on the public server at usegalaxy.org. For discussion of local Galaxy instances and the Galaxy source code, please use the Galaxy Development list: http://lists.bx.psu.edu/listinfo/galaxy-dev To manage your subscriptions to this and other Galaxy lists, please use the interface at: http://lists.bx.psu.edu/

ADD COMMENT • link written 7.8 years ago by Hiram Clawson • 260

7.8 years ago by

Loraine, Ann • 150

United States

Loraine, Ann • 150 wrote:

Hello, I think I know the answer (sort of) to this question. This may be because newer versions of tophat stopped running the "wiggles" program, which is still part of the tophat distribution and is the program that makes the "coverage.wig" file. A later version of tophat might bring this back, however - there's a note to this effect in the tophat python code. So if you can run wiggles, you can make the "coverage.wig" file on your own. A student here at UNC Charlotte (Adam Baxter) made a few changes to the "wiggles" source code that would allow you to use it with samtools to make a "coverage.wig" file from the "accepted_hits.bam" file that TopHat creates. If you (or anyone else) would like a copy, please email Adam, who is cc'ed on this email. We would be happy to help add it to Galaxy if this would be of interest to you or other Galaxy users. If there is any way we can be of assistance, please let us know! Very best wishes, Ann Loraine -- Ann Loraine Associate Professor Dept. of Bioinformatics and Genomics, UNCC North Carolina Research Campus 600 Laureate Way Kannapolis, NC 28081 704-250-5750 www.transvar.org

ADD COMMENT • link written 7.8 years ago by Loraine, Ann • 150

Hi all, Ann is correct - Tophat does not produce .wig files when run anymore. However, it's fairly easy to use Galaxy to make a wiggle-like coverage file from a BAM file: (a) run the pileup tool on your BAM to create a pileup file; (b) cut columns 1 and 4 to get your coverage file. A final note: it's often difficult to visualize coverage files because they're so large. You might be better off visualizing the BAM file and using the coverage file for statistics. Best, J.

ADD REPLY • link written 7.8 years ago by Jeremy Goecks • 2.2k

Hi, You can get an equivalent visualisation from the IGV viewer by the Broad Institute - its under IGV tools and generates a tdf file from bam or sam files. This also gives a quick and easy way of looking at depth at any particular site and is very accessible. Cheers David

ADD REPLY • link written 7.8 years ago by David Matthews • 630

I'm a fan of viewing "coverage.wig" files - they give a nice summary of read distribution over a chromosome. What I like about the "coverage.wig" file is that it captures the full read depth at every base. TDF does something different - it summarizes over (relatively) large regions. (But this can be useful, too!) If you use Integrated Genome Browser bioviz.org/igb you can open the "coverage.wig" file and view it directly. I'm a bit biased of course -- my lab contributes to IGB -- but I think you would like how it handles "wig" and short read alignment "bam" files. I wrote some documentation about "wig" files on the IGB Users Guide Web site. If you have any questions, let us know - we are happy to help: Opening "wig" files in IGB https://wiki.transvar.org/confluence/x/w4BJAQ Galaxy is a such an accessible resource -- we are trying hard to make IGB (and all the tools we develop!) as easy to use and intuitive as Galaxy. Very best wishes, Ann -- Ann Loraine Associate Professor Dept. of Bioinformatics and Genomics, UNCC North Carolina Research Campus 600 Laureate Way Kannapolis, NC 28081 704-250-5750 www.transvar.org

ADD REPLY • link written 7.8 years ago by Loraine, Ann • 150

Hi, I am not a computer person and very much like using Galaxy because its nice and easy for non-bioinformaticians. Tomorrow I am going to have a meeting with the computer science department here at Bristol and I am hoping to persuade them to install Galaxy within the High Performance Computing Centre for University of Bristol users. As I understand it this is relatively straight forward - that is to say the project was designed for them to install the whole thing locally and reproduce the set up here so I and others can use it here instead of clogging up your machines with our data and requests (!). Is this right? There are no fees or anything and this is something most computer centres should be able to do? I know this may seem a silly question but it seems prudent to ask! Cheers David

ADD REPLY • link written 7.8 years ago by David Matthews • 630

Hi David, Yes, it is free and easy to set up, and we have instructions here: https://bitbucket.org/galaxy/galaxy-central/wiki/GetGalaxy Thanks! Kanwei On Mon, Feb 21, 2011 at 5:42 PM, David Matthews

ADD REPLY • link written 7.8 years ago by Kanwei Li • 270

You may also want to point the tech folks at the following page: https://bitbucket.org/galaxy/galaxy- central/wiki/Config/ProductionServer It's linked at the bottom of the page that Kanwei posted, but it's worth specific mention since it explains all of the requirements for setting up a Galaxy server for multi-user, cluster, etc. use. --nate

ADD REPLY • link written 7.8 years ago by Nate Coraor ♦ 3.2k

Dear Ann and Jeremy: We have this discussion long time ago, and I am sorry that I brought it up here again. I am just thinking that as Ann said, can we add this tool which convert bam into wig file into galaxy? Or make a workflow to generate a wig file from a bam file generate by tophat? In this way we can just easily get a wig file from galaxy and will be able to see it in IGB. I know this may seems unnecessary for the purpose of statistical analysis, but if we can see the coverage with IGB, sometimes it is helpful to pick up interesting points quickly for specific genes. This may seems a old fashion way but my boss is a big fan of using IGB to see expression file(wig or sgr file) and do some analysis. THanks a lot! BEst Ying Quoting Jeremy Goecks <jeremy.goecks@emory.edu>: Ying Zhang, M.D., Ph.D. Postdoctoral Associate Department of Genetics, Yale University School of Medicine 300 Cedar Street,S320 New Haven, CT 06519 Tel: (203)737-2616 Fax: (203)737-2286

ADD REPLY • link written 7.6 years ago by Ying Zhang • 70

I am not sure about IGB but IGV can take directly Bam files from Tophat output, so should not be an issue of visualization. Vasu Subject: Re: [galaxy-user] get wig file after tophat To: "Jeremy Goecks" <jeremy.goecks@emory.edu> Cc: "Baxter, Adam" <adam.baxter@uncc.edu>, "galaxy-user@bx.psu.edu" <galaxy-user@bx.psu.edu> Date: Wednesday, April 20, 2011, 4:16 PM Dear Ann and Jeremy: We have this discussion long time ago, and I am sorry that I brought it up here again. I am just thinking that as Ann said, can we add this tool which convert bam into wig file into galaxy? Or make a workflow to generate a wig file from a bam file generate by tophat? In this way we can just easily get a wig file from galaxy and will be able to see it in IGB. I know this may seems unnecessary for the purpose of statistical analysis, but if we can see the coverage with IGB, sometimes it is helpful to pick up interesting points quickly for specific genes. This may seems a old fashion way but my boss is a big fan of using IGB to see expression file(wig or sgr file) and do some analysis. THanks a lot! BEst Ying Quoting Jeremy Goecks <jeremy.goecks@emory.edu>: Ying Zhang, M.D., Ph.D. Postdoctoral Associate Department of Genetics, Yale University School of Medicine 300 Cedar Street,S320 New Haven, CT 06519 Tel: (203)737-2616 Fax: (203)737-2286 ___________________________________________________________ The Galaxy User list should be used for the discussion of Galaxy analysis and other features on the public server at usegalaxy.org. Please keep all replies on the list by using "reply all" in your mail client. For discussion of local Galaxy instances and the Galaxy source code, please use the Galaxy Development list: http://lists.bx.psu.edu/listinfo/galaxy-dev To manage your subscriptions to this and other Galaxy lists, please use the interface at: http://lists.bx.psu.edu/

ADD REPLY • link written 7.6 years ago by vasu punj • 360

Thank you for the reminder and the encouraging words! We just released IGB 6.5 (many improvements!) and are planning what to do next. Top on the list is to do a better job of supporting Galaxy users by making it possible to view your "BAM" files in IGB without having to download them first. I will also investigate the BAM-to-wig issue. As we discussed previously, in our lab, we make "wig" files (locally) using the "wiggles" program from the TopHat distribution. No doubt there are other approaches that would work even better. Yours, Ann -- Ann Loraine Associate Professor Dept. of Bioinformatics and Genomics, UNCC North Carolina Research Campus 600 Laureate Way Kannapolis, NC 28081 704-250-5750 www.transvar.org

ADD REPLY • link written 7.6 years ago by Loraine, Ann • 150

Hi Ying, You're in luck because I've been working with genome browsers lately, so I think I can help you address your problem. What you're looking for is a visualization of a coverage histogram for the BAM reads produced by Tophat, yes? It turns out that some genome browsers provide this automatically as part of their solution for visualizing BAM files b/c BAM files tend to be very large and hence visualizing aggregated data is often the best solution. Both IGV and the Galaxy Trackster Browser support this functionality. I think you'll have to do some simple file conversions to get the display you want in IGV; you can check out the IGV documentation or perhaps Jim can help. I'm not sure if IGB supports this visualization mode for BAM; Ann can chime in with additional information. The Galaxy Track Browser supports coverage histograms when viewing large regions. When zoomed in, the reads are typically displayed individually, although there is a (very beta) option to create a histogram for the visible set of reads; this option may not work well (yet!) as Tophat reads often have large gaps. The top track in this visualization shows a coverage histogram for a set of Tophat reads: http://test.g2.bx.psu.edu/u/jeremy.goecks/v/assembly-of-h1-hesc-rna- seq-data Please see my previous email to Vasu for details about setting up a visualization in the Galaxy Track Browser. Best, J.

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

I can answer IGV questions, sadly I'm still coming up to speed on Galaxy. I've lost track of the original question, but IGV computes a coverage histogram on the fly, a bit like the Galaxy Track Browser, but you have to be zoomed in. However, you can also precompute a coverage histogram for the whole genome with "igvtools", a command line package. Its in a binary format (tdf) that can support viewing at any resolution in IGV. Finally, you can use igvtools to compute this as a standard "wig" file, just supply ".wig" as the extension instead of ".tdf". This is not as efficient as TDF but you can use it in other browsers, such as Galaxy and IGB. Best, Jim

ADD REPLY • link written 7.6 years ago by Jim Robinson • 150

7.8 years ago by

Jeremy Goecks • 2.2k

Jeremy Goecks • 2.2k wrote:

Ying, You can create a coverage file from a BAM file using this workflow: http://main.g2.bx.psu.edu/u/jeremy/w/create-coverage-dataset-from-bam- dataset While this doesn't generate a proper wiggle file, it can be a useful file for statistical analysis. I and others have suggested ways to visualize a BAM file with coverage data in mind: Trackster, IGV, and IGB. Thanks, J.

ADD COMMENT • link written 7.8 years ago by Jeremy Goecks • 2.2k

Ying and all; Nice discussion and lots of great ideas. One other approach is to generate bigWig files. You can serve these directly from Galaxy and they have the same advantages as BAM files; they are compressed and individual regions that be fetched on demand by UCSC. This tool in the community tool shed creates bigWig coverage files directly from BAM alignments: http://bit.ly/fxnUJ3 Brad

ADD REPLY • link written 7.8 years ago by Brad Chapman • 240

7.8 years ago by

David Matthews • 630

United Kingdom

David Matthews • 630 wrote:

HI, The option you need in IGV tools is "count". You set a window size and this gives you a tdf file from your sorted bam (or sam) file which is nice and quick to view on IGV. 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 7.8 years ago by David Matthews • 630

David, I have been also uisng the IGV like you have suggested. Vasu Subject: Re: [galaxy-user] get wig file after tophat To: "Ying Zhang" <ying.zhang.yz323@yale.edu> Cc: "Baxter, Adam" <adam.baxter@uncc.edu>, galaxy-user@bx.psu.edu Date: Tuesday, February 22, 2011, 10:54 AM HI, The option you need in IGV tools is "count". You set a window size and this gives you a tdf file from your sorted bam (or sam) file which is nice and quick to view on IGV. 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 Dear David: thank you very much for helping me! I have download the IGV and I do find the IGVtools, however, I am not sure which tool I should use for generate a tdf file, the tile function will generate a tdf file, but the input file format does not include bam or sam file, instead it need wig file. But I have no wig file to put in. So I am wondering whether you need to use other tool first. I really appreciate your help! Thank you very much! Best Ying Quoting David Matthews <d.a.matthews@bristol.ac.uk>: Hi, You can get an equivalent visualisation from the IGV viewer by the Broad Institute - its under IGV tools and generates a tdf file from bam or sam files. This also gives a quick and easy way of looking at depth at any particular site and is very accessible. Cheers David Hi all, Ann is correct - Tophat does not produce .wig files when run anymore. However, it's fairly easy to use Galaxy to make a wiggle-like coverage file from a BAM file: (a) run the pileup tool on your BAM to create a pileup file; (b) cut columns 1 and 4 to get your coverage file. A final note: it's often difficult to visualize coverage files because they're so large. You might be better off visualizing the BAM file and using the coverage file for statistics. Best, J. Hello, I think I know the answer (sort of) to this question. This may be because newer versions of tophat stopped running the "wiggles" program, which is still part of the tophat distribution and is the program that makes the "coverage.wig" file. A later version of tophat might bring this back, however - there's a note to this effect in the tophat python code. So if you can run wiggles, you can make the "coverage.wig" file on your own. A student here at UNC Charlotte (Adam Baxter) made a few changes to the "wiggles" source code that would allow you to use it with samtools to make a "coverage.wig" file from the "accepted_hits.bam" file that TopHat creates. If you (or anyone else) would like a copy, please email Adam, who is cc'ed on this email. We would be happy to help add it to Galaxy if this would be of interest to you or other Galaxy users. If there is any way we can be of assistance, please let us know! Very best wishes, Ann Loraine Hi: I am using tophat in galaxy to analyze my paired-end RNA-seq data and find out that after the tophat analysis, we can not get the wig file from it anymore which is used to be able to. Do you have any idea of how to still be able to get the wig file after tophat analysis? Thanks a lot! Best Ying Zhang, M.D., Ph.D. Postdoctoral Associate Department of Genetics, Yale University School of Medicine 300 Cedar Street,S320 New Haven, CT 06519 Tel: (203)737-2616 Fax: (203)737-2286 _______________________________________________ The Galaxy User list should be used for the discussion of Galaxy analysis and other features on the public server at usegalaxy.org. For discussion of local Galaxy instances and the Galaxy source code, please use the Galaxy Development list: http://lists.bx.psu.edu/listinfo/galaxy-dev To manage your subscriptions to this and other Galaxy lists, please use the interface at: http://lists.bx.psu.edu/ -- Ann Loraine Associate Professor Dept. of Bioinformatics and Genomics, UNCC North Carolina Research Campus 600 Laureate Way Kannapolis, NC 28081 704-250-5750 www.transvar.org _______________________________________________ The Galaxy User list should be used for the discussion of Galaxy analysis and other features on the public server at usegalaxy.org. For discussion of local Galaxy instances and the Galaxy source code, please use the Galaxy Development list: http://lists.bx.psu.edu/listinfo/galaxy-dev To manage your subscriptions to this and other Galaxy lists, please use the interface at: http://lists.bx.psu.edu/ _______________________________________________ The Galaxy User list should be used for the discussion of Galaxy analysis and other features on the public server at usegalaxy.org. For discussion of local Galaxy instances and the Galaxy source code, please use the Galaxy Development list: http://lists.bx.psu.edu/listinfo/galaxy-dev To manage your subscriptions to this and other Galaxy lists, please use the interface at: http://lists.bx.psu.edu/ _______________________________________________ The Galaxy User list should be used for the discussion of Galaxy analysis and other features on the public server at usegalaxy.org. For discussion of local Galaxy instances and the Galaxy source code, please use the Galaxy Development list: http://lists.bx.psu.edu/listinfo/galaxy-dev To manage your subscriptions to this and other Galaxy lists, please use the interface at: http://lists.bx.psu.edu/ Ying Zhang, M.D., Ph.D. Postdoctoral Associate Department of Genetics, Yale University School of Medicine 300 Cedar Street,S320 New Haven, CT 06519 Tel: (203)737-2616 Fax: (203)737-2286 _______________________________________________ The Galaxy User list should be used for the discussion of Galaxy analysis and other features on the public server at usegalaxy.org. For discussion of local Galaxy instances and the Galaxy source code, please use the Galaxy Development list: http://lists.bx.psu.edu/listinfo/galaxy-dev To manage your subscriptions to this and other Galaxy lists, please use the interface at: http://lists.bx.psu.edu/

ADD REPLY • link written 7.8 years ago by vasu punj • 360

All, For visualization, Galaxy now provides a built-in browser called Trackster. Trackster can visualize BAM files--as well as the junction file produced by Tophat and the GFF files produced by Cufflinks--and also provides coverage/summary information for all datatypes (see attached image). You can start using Trackster by going to the Visualization --> New Track Browser to set up a browser and add tracks. Alternatively, you can click on the Trackster icon in a dataset to visualize the dataset in a new or existing visualization (see attached image). Thanks, J.

ADD REPLY • link written 7.8 years ago by Jeremy Goecks • 2.2k

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