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

On 2/21/11 3:39 PM, "Ying Zhang" ying.zhang.yz323@yale.edu 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 _______________________________________________ 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/

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

On 2/21/11 5:35 PM, "David Matthews" D.A.Matthews@bristol.ac.uk wrote:

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

On 21 Feb 2011, at 21:44, Jeremy Goecks wrote:

...

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

On 2/21/11 3:39 PM, "Ying Zhang" ying.zhang.yz323@yale.edu 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 _______________________________________________ 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/

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 D.A.Matthews@bristol.ac.ukwrote:

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

---

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:

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To manage your subscriptions to this and other Galaxy lists, please use the interface at:

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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:

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

On 2/21/11 3:39 PM, "Ying Zhang" ying.zhang.yz323@yale.edu 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 _______________________________________________ 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/

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

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

On 4/20/11 5:16 PM, "Ying Zhang" ying.zhang.yz323@yale.edu wrote:

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:

...

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

On 2/21/11 3:39 PM, "Ying Zhang" ying.zhang.yz323@yale.edu 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 _______________________________________________ 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/

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

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

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.

...

On 4/20/11 5:16 PM, "Ying Zhang" ying.zhang.yz323@yale.edu wrote:

...

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:

...

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

On 2/21/11 3:39 PM, "Ying Zhang" ying.zhang.yz323@yale.edu 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 _______________________________________________ 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/

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

-- 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. 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/