RNA seq analysis

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RNA seq analysis

puvan001
Hi

I have a couple of questions regarding RNA seq analysis. My questions are
1.I need to use a viral genome (very small, ~2kb ) as a reference genome
and it is not available in Galaxy. I guess I can use this data from my
history. I have a fasta file but I am not sure whether I have to do some
kind of indexing or not.

2. In Tophat, default for "maximum number of alignments to be allowed" is
40. What my understanding is a single read can be aligned maximum 40
different places. I am wondering why this is 40. Is there any specific
reason? If I need unique mapping, I have to use 1 instead of 40. Am I
correct?


Thanks

SP


 

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Re: RNA seq analysis

David Matthews
Hi,

I have done exactly the same kind of thing for adenovirus so I can help with it. In answer to question 1 you do not need to index it will be done for you when tophat is called. Secondly you should leave the 40 multihits as it is and post analysis filter out the multihits - this will allow you to determine if you do have a multihit problem or not and if so whether it is a big problem and where it is on the genome. I have a workflow on Galaxy which you can use called "Bristol workflow to get sorted unique proper pair mapped reads". If you plug in your sam file it should give you files listing only unique hits and those which map more than once. This workflow assumes you have paired end data but it can be modified to work with single end reads as well.

Hope this helps.


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







On 6 May 2011, at 17:09, [hidden email] wrote:

Hi

I have a couple of questions regarding RNA seq analysis. My questions are
1.I need to use a viral genome (very small, ~2kb ) as a reference genome and it is not available in Galaxy. I guess I can use this data from my history. I have a fasta file but I am not sure whether I have to do some kind of indexing or not.

2. In Tophat, default for "maximum number of alignments to be allowed" is 40. What my understanding is a single read can be aligned maximum 40 different places. I am wondering why this is 40. Is there any specific reason? If I need unique mapping, I have to use 1 instead of 40. Am I correct?


Thanks

SP



___________________________________________________________
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/


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Re: RNA seq analysis

puvan001

Hi David,

Thanks!When I tried to run Tophat, it doesn't recognise my FASTA file and
it says "History does not include a dataset of the required format /
build". Do you have any thoughts about this?

Now it makes more sense about "multihits". Thanks for sharing your
workflow.

With regards

Sumathy

On May 6 2011, David Matthews wrote:

>Hi,
>
 I have done exactly the same kind of thing for adenovirus so I can help
with it. In answer to question 1 you do not need to index it will be done
for you when tophat is called. Secondly you should leave the 40 multihits
as it is and post analysis filter out the multihits - this will allow you
to determine if you do have a multihit problem or not and if so whether it
is a big problem and where it is on the genome. I have a workflow on Galaxy
which you can use called "Bristol workflow to get sorted unique proper pair
mapped reads". If you plug in your sam file it should give you files
listing only unique hits and those which map more than once. This workflow
assumes you have paired end data but it can be modified to work with single
end reads as well.

>
>Hope this helps.
>
>
>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
>
>[hidden email]
>
>
>
>
>
>
>On 6 May 2011, at 17:09, [hidden email] wrote:
>
>> Hi
>>
>> I have a couple of questions regarding RNA seq analysis. My questions are
   1.I need to use a viral genome (very small, ~2kb ) as a reference genome
and it is not available in Galaxy. I guess I can use this data from my
history. I have a fasta file but I am not sure whether I have to do some
kind of indexing or not.
>>
   2. In Tophat, default for "maximum number of alignments to be allowed"
is 40. What my understanding is a single read can be aligned maximum 40
different places. I am wondering why this is 40. Is there any specific
reason? If I need unique mapping, I have to use 1 instead of 40. Am I
correct?

>>
>>
>> Thanks
>>
>> SP
>>
>>
>>
>> ___________________________________________________________
>> 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/
>
>

--
Sumathy Puvanendiran
Graduate student



___________________________________________________________
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Galaxy analysis and other features on the public server
at usegalaxy.org.  Please keep all replies on the list by
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Re: RNA seq analysis

Austin Paul
Hi,
 
You need to run fastq groomer on your rna-seq data.  Your reference is fine as a fasta.
 
Austin

On Fri, May 6, 2011 at 10:26 AM, <[hidden email]> wrote:

Hi David,

Thanks!When I tried to run Tophat, it doesn't recognise my FASTA file and it says "History does not include a dataset of the required format / build". Do you have any thoughts about this?

Now it makes more sense about "multihits". Thanks for sharing your workflow.

With regards

Sumathy


On May 6 2011, David Matthews wrote:

Hi,

I have done exactly the same kind of thing for adenovirus so I can help with it. In answer to question 1 you do not need to index it will be done for you when tophat is called. Secondly you should leave the 40 multihits as it is and post analysis filter out the multihits - this will allow you to determine if you do have a multihit problem or not and if so whether it is a big problem and where it is on the genome. I have a workflow on Galaxy which you can use called "Bristol workflow to get sorted unique proper pair mapped reads". If you plug in your sam file it should give you files listing only unique hits and those which map more than once. This workflow assumes you have paired end data but it can be modified to work with single end reads as well.

Hope this helps.


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

[hidden email]






On 6 May 2011, at 17:09, [hidden email] wrote:

Hi

I have a couple of questions regarding RNA seq analysis. My questions are
 1.I need to use a viral genome (very small, ~2kb ) as a reference genome and it is not available in Galaxy. I guess I can use this data from my history. I have a fasta file but I am not sure whether I have to do some kind of indexing or not.

 2. In Tophat, default for "maximum number of alignments to be allowed" is 40. What my understanding is a single read can be aligned maximum 40 different places. I am wondering why this is 40. Is there any specific reason? If I need unique mapping, I have to use 1 instead of 40. Am I correct?


Thanks

SP



___________________________________________________________
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/



--
Sumathy Puvanendiran
Graduate student




___________________________________________________________
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
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use the Galaxy Development list:

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

 http://lists.bx.psu.edu/


___________________________________________________________
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Galaxy analysis and other features on the public server
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Re: RNA seq analysis

puvan001

Hi Austin

 I did all these (grooming and trimming)on rna-seq data and I don't have a
problem with built in genome . I'll try again!


Thanks

Sumathy




On May 6 2011, Austin Paul wrote:

>Hi,
>
>You need to run fastq groomer on your rna-seq data.  Your reference is fine
>as a fasta.
>
>Austin
>
>On Fri, May 6, 2011 at 10:26 AM, <[hidden email]> wrote:
>
>>
>> Hi David,
>>
>> Thanks!When I tried to run Tophat, it doesn't recognise my FASTA file and
   it says "History does not include a dataset of the required format /
build".

>> Do you have any thoughts about this?
>>
>> Now it makes more sense about "multihits". Thanks for sharing your
>> workflow.
>>
>> With regards
>>
>> Sumathy
>>
>>
>> On May 6 2011, David Matthews wrote:
>>
>> Hi,
>>>
>>> I have done exactly the same kind of thing for adenovirus so I can help
>> with it. In answer to question 1 you do not need to index it will be done
   for you when tophat is called. Secondly you should leave the 40
multihits as
>> it is and post analysis filter out the multihits - this will allow you to
   determine if you do have a multihit problem or not and if so whether it
is a
   big problem and where it is on the genome. I have a workflow on Galaxy
which
   you can use called "Bristol workflow to get sorted unique proper pair
mapped
   reads". If you plug in your sam file it should give you files listing
only
>> unique hits and those which map more than once. This workflow assumes you
   have paired end data but it can be modified to work with single end
reads as

>> well.
>>
>>>
>>> Hope this helps.
>>>
>>>
>>> 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
>>>
>>> [hidden email]
>>>
>>>
>>>
>>>
>>>
>>>
>>> On 6 May 2011, at 17:09, [hidden email] wrote:
>>>
>>> Hi
>>>>
     I have a couple of questions regarding RNA seq analysis. My questions
are
>>>>
     1.I need to use a viral genome (very small, ~2kb ) as a reference
genome

>> and it is not available in Galaxy. I guess I can use this data from my
>> history. I have a fasta file but I am not sure whether I have to do some
>> kind of indexing or not.
>>
>>>
>>>>  2. In Tophat, default for "maximum number of alignments to be allowed"
>> is 40. What my understanding is a single read can be aligned maximum 40
>> different places. I am wondering why this is 40. Is there any specific
>> reason? If I need unique mapping, I have to use 1 instead of 40. Am I
>> correct?
>>
>>>
>>>>
>>>> Thanks
>>>>
>>>> SP
>>>>
>>>>
>>>>
>>>> ___________________________________________________________
>>>> 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/
>>>>
>>>
>>>
>>>
>> --
>> Sumathy Puvanendiran
>> Graduate student
>>
>>
>>
>>
>> ___________________________________________________________
>> 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/
>>
>

--
Sumathy Puvanendiran
Graduate student


___________________________________________________________
The Galaxy User list should be used for the discussion of
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Re: RNA seq analysis

puvan001
In reply to this post by David Matthews
Hello

I was able to run RNA seq data against a custom build genome. How can I
visualize the results. I tried via trackster and unfortunately I couldn't.
Can you help me?


Thanks

Sumathy
___________________________________________________________
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Re: RNA seq analysis

Austin Paul
There are many ways.  I typically use IGV.  It needs a sam file, so I first convert the bam to sam in galaxy, then download the sam file.  In IGV, I upload the reference and the sam file, then use IGVtools to index the sam file, then I can visualize the data.
 
Austin
On Fri, May 6, 2011 at 5:30 PM, <[hidden email]> wrote:
Hello

I was able to run RNA seq data against a custom build genome. How can I visualize the results. I tried via trackster and unfortunately I couldn't. Can you help me?


Thanks

Sumathy

___________________________________________________________
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Re: RNA seq analysis

Sean Davis
IGV reads BAM files just fine; no need to convert to SAM.

Sean

On Fri, May 6, 2011 at 8:45 PM, Austin Paul <[hidden email]> wrote:
There are many ways.  I typically use IGV.  It needs a sam file, so I first convert the bam to sam in galaxy, then download the sam file.  In IGV, I upload the reference and the sam file, then use IGVtools to index the sam file, then I can visualize the data.
 
Austin
On Fri, May 6, 2011 at 5:30 PM, <[hidden email]> wrote:
Hello

I was able to run RNA seq data against a custom build genome. How can I visualize the results. I tried via trackster and unfortunately I couldn't. Can you help me?


Thanks

Sumathy

___________________________________________________________
The Galaxy User list should be used for the discussion of
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Re: RNA seq analysis

Austin Paul
Oops.  Good to know.  Thanks.
 
Austin

On Fri, May 6, 2011 at 6:02 PM, Sean Davis <[hidden email]> wrote:
IGV reads BAM files just fine; no need to convert to SAM.

Sean

On Fri, May 6, 2011 at 8:45 PM, Austin Paul <[hidden email]> wrote:
There are many ways.  I typically use IGV.  It needs a sam file, so I first convert the bam to sam in galaxy, then download the sam file.  In IGV, I upload the reference and the sam file, then use IGVtools to index the sam file, then I can visualize the data.
 
Austin
On Fri, May 6, 2011 at 5:30 PM, <[hidden email]> wrote:
Hello

I was able to run RNA seq data against a custom build genome. How can I visualize the results. I tried via trackster and unfortunately I couldn't. Can you help me?


Thanks

Sumathy

___________________________________________________________
The Galaxy User list should be used for the discussion of
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Re: RNA seq analysis

vasu punj
In reply to this post by puvan001
I generally take the GTF file to UCSC genome browser.
If you are visualizing Bam file after alignment. I found IGV convinenet, though you may be able to visualize in Galaxy.
 
Vasu

--- On Fri, 5/6/11, [hidden email] <[hidden email]> wrote:

From: [hidden email] <[hidden email]>
Subject: Re: [galaxy-user] RNA seq analysis
To: "David Matthews" <[hidden email]>
Cc: [hidden email]
Date: Friday, May 6, 2011, 7:30 PM

Hello

I was able to run RNA seq data against a custom build genome. How can I visualize the results. I tried via trackster and unfortunately I couldn't. Can you help me?


Thanks

Sumathy
___________________________________________________________
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Re: RNA seq analysis

vasu punj
In reply to this post by Sean Davis
One of the problem is IGV dont have option of creating index file so one has to create index file in Galaxy first to  view in IGV. Jim I have been using IGV 2 beta version it is great work but How hard is to include index functionality with in IGV. I know we can use sam tools also but just for convinence if it is not that much of work.
Vasu

--- On Fri, 5/6/11, Sean Davis <[hidden email]> wrote:

From: Sean Davis <[hidden email]>
Subject: Re: [galaxy-user] RNA seq analysis
To: "Austin Paul" <[hidden email]>
Cc: "[hidden email]" <[hidden email]>, "[hidden email]" <[hidden email]>
Date: Friday, May 6, 2011, 8:02 PM

IGV reads BAM files just fine; no need to convert to SAM.
Sean

On Fri, May 6, 2011 at 8:45 PM, Austin Paul <austinpa@...> wrote:
There are many ways.  I typically use IGV.  It needs a sam file, so I first convert the bam to sam in galaxy, then download the sam file.  In IGV, I upload the reference and the sam file, then use IGVtools to index the sam file, then I can visualize the data.
 
Austin
On Fri, May 6, 2011 at 5:30 PM, <puvan001@...> wrote:
Hello

I was able to run RNA seq data against a custom build genome. How can I visualize the results. I tried via trackster and unfortunately I couldn't. Can you help me?


Thanks

Sumathy

___________________________________________________________
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-----Inline Attachment Follows-----

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Re: RNA seq analysis

Jeremy Goecks
In reply to this post by puvan001
Sumathy,

What kind of problems are you having with Trackster?

J.

On May 6, 2011, at 8:30 PM, <[hidden email]> wrote:

> Hello
>
> I was able to run RNA seq data against a custom build genome. How can I visualize the results. I tried via trackster and unfortunately I couldn't. Can you help me?
>
>
> Thanks
>
> Sumathy
> ___________________________________________________________
> 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/


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Re: RNA seq analysis

Jim Robinson
In reply to this post by vasu punj
Hi Vasu,

I'm going to add the function to index BAM files soon, using Picard.    
In the beginning....  there was no java BAM reader, only SAM, and I  
added the index then.  Indexed BAMs came along later, but that's  
probably more than you want to know...    I think most people will  
still use Galaxy to index as it can take a long time, but I agree with  
you on the convenience factor.

Jim


On May 6, 2011, at 9:36 PM, vasu punj wrote:

> One of the problem is IGV dont have option of creating index file so  
> one has to create index file in Galaxy first to  view in IGV. Jim I  
> have been using IGV 2 beta version it is great work but How hard is  
> to include index functionality with in IGV. I know we can use sam  
> tools also but just for convinence if it is not that much of work.
> Vasu
>
> --- On Fri, 5/6/11, Sean Davis <[hidden email]> wrote:
>
> From: Sean Davis <[hidden email]>
> Subject: Re: [galaxy-user] RNA seq analysis
> To: "Austin Paul" <[hidden email]>
> Cc: "[hidden email]" <[hidden email]>, "[hidden email]
> " <[hidden email]>
> Date: Friday, May 6, 2011, 8:02 PM
>
> IGV reads BAM files just fine; no need to convert to SAM.
> Sean
>
> On Fri, May 6, 2011 at 8:45 PM, Austin Paul <[hidden email]> wrote:
> There are many ways.  I typically use IGV.  It needs a sam file, so  
> I first convert the bam to sam in galaxy, then download the sam  
> file.  In IGV, I upload the reference and the sam file, then use  
> IGVtools to index the sam file, then I can visualize the data.
>
> Austin
> On Fri, May 6, 2011 at 5:30 PM, <[hidden email]> wrote:
> Hello
>
> I was able to run RNA seq data against a custom build genome. How  
> can I visualize the results. I tried via trackster and unfortunately  
> I couldn't. Can you help me?
>
>
> Thanks
>
> Sumathy
>
> ___________________________________________________________
> 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/
>
>
>
> -----Inline Attachment Follows-----
>
> ___________________________________________________________
> 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/
> ___________________________________________________________
> 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/

___________________________________________________________
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Re: RNA seq analysis

puvan001
In reply to this post by Jeremy Goecks


Hi

I may be doing in a wrong way. I clicked trackster and I added the custom
build genome. Since it is a very small genome (~2kb), I considered this as
a single contig. Then I cliked "add tracks" and added my data file. But I
got a message "no data for this contig. Whenever I used built in genomes I
did not have any problem. I guess I am doing something wrong here.


Sumathy










On May 6 2011, Jeremy Goecks wrote:

>Sumathy,
>
>What kind of problems are you having with Trackster?
>
>J.
>
>On May 6, 2011, at 8:30 PM, <[hidden email]> wrote:
>
>> Hello
>>
   I was able to run RNA seq data against a custom build genome. How can I
visualize the results. I tried via trackster and unfortunately I couldn't.
Can you help me?

>>
>>
>> Thanks
>>
>> Sumathy
>> ___________________________________________________________
>> 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/
>
>

--
Sumathy Puvanendiran
Graduate student


___________________________________________________________
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
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Re: RNA seq analysis

puvan001
In reply to this post by vasu punj

Hi

Thanks! I am little bit familiar with IGV. I'll try then.



Sumathy



On May 6 2011, vasu punj wrote:

 One of the problem is IGV dont have option of creating index file so one
has to create index file in Galaxy first to  view in IGV. Jim I have been
using IGV 2 beta version it is great work but How hard is to include index
functionality with in IGV. I know we can use sam tools also but just for
convinence if it is not that much of work.
>Vasu
>
>--- On Fri, 5/6/11, Sean Davis <[hidden email]> wrote:
>
>
>From: Sean Davis <[hidden email]>
>Subject: Re: [galaxy-user] RNA seq analysis
>To: "Austin Paul" <[hidden email]>
 Cc: "[hidden email]" <[hidden email]>,
"[hidden email]" <[hidden email]>

>Date: Friday, May 6, 2011, 8:02 PM
>
>
>IGV reads BAM files just fine; no need to convert to SAM.
>
>Sean
>
>
>On Fri, May 6, 2011 at 8:45 PM, Austin Paul <[hidden email]> wrote:
>
>
 There are many ways.  I typically use IGV.  It needs a sam file, so I
first convert the bam to sam in galaxy, then download the sam file.  In
IGV, I upload the reference and the sam file, then use IGVtools to index
the sam file, then I can visualize the data.

>Austin
>
>On Fri, May 6, 2011 at 5:30 PM, <[hidden email]> wrote:
>
>Hello
>
 I was able to run RNA seq data against a custom build genome. How can I
visualize the results. I tried via trackster and unfortunately I couldn't.
Can you help me?

>
>
>Thanks
>
>Sumathy
>
>
>
>___________________________________________________________
>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/
>
>
>
>-----Inline Attachment Follows-----
>
>
>___________________________________________________________
>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/

--
Sumathy Puvanendiran
Graduate student



___________________________________________________________
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
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Re: RNA seq analysis

vasu punj
In reply to this post by Jim Robinson
Thanks Jim,
 
Vasu
 
--- On Fri, 5/6/11, Jim Robinson <[hidden email]> wrote:

From: Jim Robinson <[hidden email]>
Subject: Re: [galaxy-user] RNA seq analysis
To: "vasu punj" <[hidden email]>
Cc: "Austin Paul" <[hidden email]>, "Sean Davis" <[hidden email]>, "[hidden email]" <[hidden email]>, "[hidden email]" <[hidden email]>
Date: Friday, May 6, 2011, 9:01 PM

Hi Vasu,

I'm going to add the function to index BAM files soon, using Picard.   In the beginning....  there was no java BAM reader, only SAM, and I added the index then.  Indexed BAMs came along later, but that's probably more than you want to know...    I think most people will still use Galaxy to index as it can take a long time, but I agree with you on the convenience factor.

Jim


On May 6, 2011, at 9:36 PM, vasu punj wrote:

> One of the problem is IGV dont have option of creating index file so one has to create index file in Galaxy first to  view in IGV. Jim I have been using IGV 2 beta version it is great work but How hard is to include index functionality with in IGV. I know we can use sam tools also but just for convinence if it is not that much of work.
> Vasu
>
> --- On Fri, 5/6/11, Sean Davis <ssdavis2@...> wrote:
>
> From: Sean Davis <sdavis2@...>
> Subject: Re: [galaxy-user] RNA seq analysis
> To: "Austin Paul" <austinpa@...>
> Cc: "galaxy-user@..." <galaxy-user@...>, "puvan001@..." <puvan001@...>
> Date: Friday, May 6, 2011, 8:02 PM
>
> IGV reads BAM files just fine; no need to convert to SAM.
> Sean
>
> On Fri, May 6, 2011 at 8:45 PM, Austin Paul <austinpa@...> wrote:
> There are many ways.  I typically use IGV.  It needs a sam file, so I first convert the bam to sam in galaxy, then download the sam file.  In IGV, I upload the reference and the sam file, then use IGVtools to index the sam file, then I can visualize the data.
>
> Austin
> On Fri, May 6, 2011 at 5:30 PM, <puvan001@...> wrote:
> Hello
>
> I was able to run RNA seq data against a custom build genome. How can I visualize the results. I tried via trackster and unfortunately I couldn't. Can you help me?
>
>
> Thanks
>
> Sumathy
>
> ___________________________________________________________
> 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/
>
>
>
> -----Inline Attachment Follows-----
>
> ___________________________________________________________
> 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/
> ___________________________________________________________
> 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/


___________________________________________________________
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
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Re: RNA seq analysis

Jeremy Goecks
In reply to this post by puvan001
Sumathy,

It sounds like you're on the right track. To visualize data for a custom build in Trackster, you need to create a custom build and use that in Trackster:

(1) using the top tabs in Galaxy, go to User --> Custom Builds;
(2) add a new build with the length info as follows:
<contig_name> <length>

Important note: you'll need to make sure that your contig name matches the one used in your fasta file. This is my best guess about what's causing problems for you.

(3) Create a Trackster visualization using the custom build and add your dataset.

Let us know if you have more questions/problems.

Thanks,
J.

On May 6, 2011, at 10:43 PM, <[hidden email]> wrote:

>
>
> Hi
>
> I may be doing in a wrong way. I clicked trackster and I added the custom build genome. Since it is a very small genome (~2kb), I considered this as a single contig. Then I cliked "add tracks" and added my data file. But I got a message "no data for this contig. Whenever I used built in genomes I did not have any problem. I guess I am doing something wrong here.
>
>
> Sumathy
>
>
>
>
>
>
>
>
>
>
> On May 6 2011, Jeremy Goecks wrote:
>
>> Sumathy,
>>
>> What kind of problems are you having with Trackster?
>>
>> J.
>>
>> On May 6, 2011, at 8:30 PM, <[hidden email]> wrote:
>>
>>> Hello
>  I was able to run RNA seq data against a custom build genome. How can I visualize the results. I tried via trackster and unfortunately I couldn't. Can you help me?
>>> Thanks
>>> Sumathy
>>> ___________________________________________________________
>>> 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/
>>
>>
>
> --
> Sumathy Puvanendiran
> Graduate student
>
>


___________________________________________________________
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
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Re: RNA seq analysis

puvan001

Hi

Thank you! yes, your guess is correct. Now it works.



Sumathy






On May 7 2011, Jeremy Goecks wrote:

>Sumathy,
>
 It sounds like you're on the right track. To visualize data for a custom
build in Trackster, you need to create a custom build and use that in
Trackster:
>
>(1) using the top tabs in Galaxy, go to User --> Custom Builds;
>(2) add a new build with the length info as follows:
><contig_name> <length>
>
 Important note: you'll need to make sure that your contig name matches the
one used in your fasta file. This is my best guess about what's causing
problems for you.
>
 (3) Create a Trackster visualization using the custom build and add your
dataset.

>
>Let us know if you have more questions/problems.
>
>Thanks,
>J.
>
>On May 6, 2011, at 10:43 PM, <[hidden email]> wrote:
>
>>
>>
>> Hi
>>
   I may be doing in a wrong way. I clicked trackster and I added the
custom build genome. Since it is a very small genome (~2kb), I considered
this as a single contig. Then I cliked "add tracks" and added my data file.
But I got a message "no data for this contig. Whenever I used built in
genomes I did not have any problem. I guess I am doing something wrong
here.

>>
>>
>> Sumathy
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> On May 6 2011, Jeremy Goecks wrote:
>>
>>> Sumathy,
>>>
>>> What kind of problems are you having with Trackster?
>>>
>>> J.
>>>
>>> On May 6, 2011, at 8:30 PM, <[hidden email]> wrote:
>>>
>>>> Hello
    I was able to run RNA seq data against a custom build genome. How can I
visualize the results. I tried via trackster and unfortunately I couldn't.
Can you help me?

>>>> Thanks
>>>> Sumathy
>>>> ___________________________________________________________
>>>> 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/
>>>
>>>
>>
>> --
>> Sumathy Puvanendiran
>> Graduate student
>>
>>
>
>

--
Sumathy Puvanendiran
Graduate student


___________________________________________________________
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
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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:

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