Re: [galaxy-user] Installing galaxy with Apache ...
Hi Neil, with your current apache configuration, you should probably see Galaxy at http://yourip/galaxy This is due to your Rewriterule /galaxy and for that you have also set proxy_prefix = /galaxy in your universe_wsgi.ini You should probably remove the empty galaxy directory under /var/www Furthermore, most of the time you will not need a directory with 777 in your apache web/documentroot.. Cheers, Jelle On Thu, Aug 23, 2012 at 8:34 AM, neil.burd...@csiro.au wrote: Hi I've installed galaxy with the default settings and it works fine. On a new Ubuntu machine I am trying to get galaxy running with Apache. But I am having problems, I've followed the documentation. After installing Apache I can put my ip address into a browser and I get message saying Apache is working fine. It displays a message from /var/www/index.html Now when I start galaxy I was assuming I would get redirected to the galaxy welcome page, but this doesn't happen and it remains at /var/www/index.html. To view the welcome page I have to add :8080 after the ip address. Is this still required? I thought that Apache would know to redirect to my distributionWhat am I doing wrong? /etc/apache2/sites-available/default-ssl and /etc/apache2/sites-available/default look like this ... DocumentRoot /var/www Directory / Options FollowSymLinks AllowOverride None /Directory Directory /var/www/ Options Indexes FollowSymLinks MultiViews AllowOverride None Order allow,deny allow from all /Directory etc ... /var/www/.htaccess RewriteEngine on RewriteRule ^/galaxy$ /galaxy/ [R] RewriteRule ^/galaxy/static/style/(.*) /home/galaxy/galaxy-dist/static/june_2007_style/blue/$1 [L] RewriteRule ^/galaxy/static/scripts/(.*) /home/galaxy/galaxy-dist/static/scripts/packed/$1 [L] RewriteRule ^/galaxy/static/(.*) /home/galaxy/galaxy-dist/static/$1 [L] RewriteRule ^/galaxy/favicon.ico /home/galaxy/galaxy-dist/static/favicon.ico [L] RewriteRule ^/galaxy/robots.txt /home/galaxy/galaxy-dist/static/robots.txt [L] RewriteRule ^/galaxy(.*) http://localhost:8080$1 [P] I created an empty 'galaxy' directory with 777 permissions on it under /var/www and universe_wsgi.ini # Define the proxy-prefix filter. [filter:proxy-prefix] use = egg:PasteDeploy#prefix prefix = /galaxy # Galaxy --- # Configuration of the Galaxy application. [app:main] filter-with = proxy-prefix cookie_path = /galaxy Thanks Neil ___ 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/
[galaxy-user] How much can I trimm my reads
Dear All, I am analysing RNA-seq datasets for the differential splicing events between cell types. My reads are 36bp long. In order to increase the quality of reads, I need to trim some nucleotides from ends. How many nucleotides can I trim? I am afraid that if I trim too much, the reliability of the alingment will be affected. Thanks in advance. Jianguang ___ 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/
[galaxy-user] What is the minimum Quality should I set for Filter FASTQ?
Dear All, I am analysing RNA-seq datasets for differential splicing events between cell types. Some of my reads contain bed nucleotides, should I run Filter FASTQ to remove these not so good reads? If I do need to, what is the Minimum Quality should I set for the Filter? Thanks. Jianguang ___ 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/
[galaxy-user] Should I use iGenomes verson of a reference GTF for Tophat?
Dear All, I am analysing RNA-seq datasets for differential splicing events between cell types. These are mouse cells. Jen suggested me to use the iGenomes version of reference GTF to take full advantage of the options in CuffDiff. My question is: should I use this iGenome version reference GTF when I run Tophat? Thanks. Jianguang ___ 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/
Re: [galaxy-user] How much can I trimm my reads
Hello Jianguang, This general protocol is also in the RNA-seq tutorial: http://main.g2.bx.psu.edu/u/jeremy/p/galaxy-rna-seq-analysis-exercise -- Understanding and QCing the reads That said, I had a sample of your data from before and I ran FastQC on it and see what you mean, the quality drops off steadily after the first 10 bases or so, then below phred+20 around the middle of the sequence (for both ends). There are a few options - 1 - Do as Ann suggests and just leave these alone and test to see what happens in TopHat. If the mapping fails, then you will know that you need to do some quality cleanup. 2 - Use the FastQC results to decide on a lower quality score boundary and trim the very worst sequences. Because of the length, yes, take care not to remove too much. As I stated, from the sample I looked at, even phred+20 would probably clip too aggressively. In general it is best to do as little manipulation as possible with expression data. Some testing on your part will be needed to identify the correct processing, and the same process will not apply to all datasets. But the general path outlined in the tutorial is a good one for what you are trying to do and should be able to address your questions. Take care, Jen Galaxy team On 8/23/12 7:40 AM, Du, Jianguang wrote: Dear All, I am analysing RNA-seq datasets for the differential splicing events between cell types. My reads are 36bp long. In order to increase the quality of reads, I need to trim some nucleotides from ends. How many nucleotides can I trim? I am afraid that if I trim too much, the reliability of the alingment will be affected. Thanks in advance. Jianguang ___ 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/ -- Jennifer Jackson http://galaxyproject.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/
Re: [galaxy-user] Installing Galaxy and Hooking into a SGE Cluster
No, no it's just internal billing within our organization. We're charging for compute time on the cluster, not for any particular software. No one is making any money off of Galaxy, it's just a way to fairly divide up the compute time available to the organization. -Greg On Thu, Aug 23, 2012 at 11:27 AM, John Jones mr.johnjo...@gmail.com wrote: Is it acceptable/legal to bill people for using the opensource tools in Galaxy? I can understand billing for server/cluster usage, perhaps even for a profit, but when I write a tool in my own time for free and then specify that people who distribute my tool may only do so non-commercially, it would make me very sad to think that people are getting around this buy charging excessive amounts in sever-usage-fees, and thus making a business out of my free work. I'm not saying that this is what you are doing Greg, and I'm sure you have your reasons, but I'm really just wondering what sort of copyright protection i would need as a bioinformatics software developer to prevent such abuse of charity. All the best, - John On 22 Aug 2012, at 21:20, mailing list margeem...@gmail.com wrote: Hi guys, I want to install Galaxy on a local server and hook it into our SGE cluster. This excellent document seems to provide a lot of the steps http://wiki.g2.bx.psu.edu/Admin/Config/Performance/Cluster however I have two questions I'm not sure about. 1. Right now users keep their data in their home directories. The jobs they run on the cluster have access to the user's home directory and use that data. How would this work with Galaxy? Can the Galaxy interface and jobs reference data in the users' home directories? Or I guess how does this work? 2. Also can I integrate the Galaxy user system with LDAP? And can that user information be passed to the cluster to run the jobs under that user? We need this for cluster billing purposes. Thanks in advance for any help. Everyone is our company is really pining for a Galaxy install and I'm really hoping I can set it up for them. -Greg ___ 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/
Re: [galaxy-user] Should I use iGenomes verson of a reference GTF for Tophat?
Hi Jen, Thanks for your help. Do you mean that if I want to find novel isoform/splicing, I need to select No under Use Reference Annotation when I run Cufflink, and then use iGenome version of reference GTF when I run Cuffmerge? Based on your information and some protocols found online, my understanding is that: 1) if use iGenome version of reference GTF, I only need to run Cuffmerge with the Cufflink ouputs, because iGenome version reference GTF already contains attributes such as p_id and tss_id. Then the Cuffmerge output can be used for Cuffdiff. 2) however, if I use the reference GTF from Ensembl/UCSC (rather than from iGenome), I need to run Cuffcompare to create p_id and tss_id, which is required for Cuffdiff. Am I right? Another question is: should I use iGenome version of reference GTF when I run Tophat if I want to see novel isoforms/splicing? Thanks. Jianguang From: Jennifer Jackson [j...@bx.psu.edu] Sent: Thursday, August 23, 2012 11:46 AM To: Du, Jianguang Cc: galaxy-user@lists.bx.psu.edu Subject: Re: [galaxy-user] Should I use iGenomes verson of a reference GTF for Tophat? Hello Jianguang, When in the analysis process to start using the reference GTF file can depend on whether or not you intend to do any discovery along with differential expression testing. At the TopHat and Cufflinks steps, using reference GTF file can influence how datasets will map and assemble. In general, if your intention is to do discovery (e.g. work with novel isoforms in your data, but not in the reference), then do not add in the reference GTF until the CuffMerge step (to produce the input annotation GTF file for Cuffdiff). But if you want to guide the analysis toward known isoforms, then use the reference GTF. This is the process our RNA-seq example protocol follows: http://main.g2.bx.psu.edu/u/jeremy/p/galaxy-rna-seq-analysis-exercise For reference, there are other variations of this on the Cufflinks web site, some that never lead to Cuffdiff, but still may be useful to review. Please see the Cufflinks paper (linked from right side bar as Protocol for many more options/discussion. http://cufflinks.cbcb.umd.edu/tutorial.html -- Common uses of the Cufflinks package The end decision will be up to you, and a few runs with different options may be a useful way to make the final call, but hopefully this provides some resources to help you understand the option, Jen Galaxy team On 8/23/12 8:03 AM, Du, Jianguang wrote: Dear All, I am analysing RNA-seq datasets for differential splicing events between cell types. These are mouse cells. Jen suggested me to use the iGenomes version of reference GTF to take full advantage of the options in CuffDiff. My question is: should I use this iGenome version reference GTF when I run Tophat? Thanks. Jianguang ___ 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/ -- Jennifer Jackson http://galaxyproject.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/
Re: [galaxy-user] Installing Galaxy and Hooking into a SGE Cluster
I completely understand what you're saying Greg and I have no doubt that your billing only covers the maintainence costs of your cluster - and I don't want to derail your thread because you ask a very legitimate question (to which im afraid I dont know the answer to, and would also be interested to hear the outcome of!); but I was just curious to hear if anyone in the community knew what protection there is for the developers of software if anyone can charge, say, $1000 for an alignment using free tools that takes only an hour to run. In retrospect I should never have brought this up within your thread, and should have asked the question in it's own right (perhaps in a mailing-list directed at developers rather than users). Anyway, all the best with your question :-) - John On 23 Aug 2012, at 19:01, mailing list margeem...@gmail.com wrote: No, no it's just internal billing within our organization. We're charging for compute time on the cluster, not for any particular software. No one is making any money off of Galaxy, it's just a way to fairly divide up the compute time available to the organization. -Greg On Thu, Aug 23, 2012 at 11:27 AM, John Jones mr.johnjo...@gmail.com wrote: Is it acceptable/legal to bill people for using the opensource tools in Galaxy? I can understand billing for server/cluster usage, perhaps even for a profit, but when I write a tool in my own time for free and then specify that people who distribute my tool may only do so non-commercially, it would make me very sad to think that people are getting around this buy charging excessive amounts in sever-usage-fees, and thus making a business out of my free work. I'm not saying that this is what you are doing Greg, and I'm sure you have your reasons, but I'm really just wondering what sort of copyright protection i would need as a bioinformatics software developer to prevent such abuse of charity. All the best, - John On 22 Aug 2012, at 21:20, mailing list margeem...@gmail.com wrote: Hi guys, I want to install Galaxy on a local server and hook it into our SGE cluster. This excellent document seems to provide a lot of the steps http://wiki.g2.bx.psu.edu/Admin/Config/Performance/Cluster however I have two questions I'm not sure about. 1. Right now users keep their data in their home directories. The jobs they run on the cluster have access to the user's home directory and use that data. How would this work with Galaxy? Can the Galaxy interface and jobs reference data in the users' home directories? Or I guess how does this work? 2. Also can I integrate the Galaxy user system with LDAP? And can that user information be passed to the cluster to run the jobs under that user? We need this for cluster billing purposes. Thanks in advance for any help. Everyone is our company is really pining for a Galaxy install and I'm really hoping I can set it up for them. -Greg ___ 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/
Re: [galaxy-user] Installing Galaxy and Hooking into a SGE Cluster
On 23 August 2012 14:39, John Jones mr.johnjo...@gmail.com wrote: [...] knew what protection there is for the developers of software if anyone can charge, say, $1000 for an alignment using free tools that takes only an hour to run. Many open source licenses will permit anyone to charge for redistributing the software and/or reselling services regarding the software. It is up to the users of the software to realize that they do not need to pay fees, which I think makes a lot of sense because the only other option would be to sue license violators. At least I cannot afford such a thing. Making money out of open source software is also not a bad thing. You can get Linux for free, or you can pay RedHat many monies for also receiving additional services. You can install Darwin for free, or you pay and get Apple's extensions on top of it. Best, Joachim ___ 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/
Re: [galaxy-user] Installing Galaxy and Hooking into a SGE Cluster
Anyway, back to my question. Does anyone know? Would I be better off asking on the developer mailing list or perhaps Stack Overflow? Thanks again, Greg On Thu, Aug 23, 2012 at 2:59 PM, Joachim Baran joachim.ba...@gmail.com wrote: On 23 August 2012 14:39, John Jones mr.johnjo...@gmail.com wrote: [...] knew what protection there is for the developers of software if anyone can charge, say, $1000 for an alignment using free tools that takes only an hour to run. Many open source licenses will permit anyone to charge for redistributing the software and/or reselling services regarding the software. It is up to the users of the software to realize that they do not need to pay fees, which I think makes a lot of sense because the only other option would be to sue license violators. At least I cannot afford such a thing. Making money out of open source software is also not a bad thing. You can get Linux for free, or you can pay RedHat many monies for also receiving additional services. You can install Darwin for free, or you pay and get Apple's extensions on top of it. Best, Joachim ___ 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/
Re: [galaxy-user] Should I use iGenomes verson of a reference GTF for Tophat?
Hello Jianguang, On 8/23/12 11:28 AM, Du, Jianguang wrote: Hi Jen, Thanks for your help. Do you mean that if I want to find novel isoform/splicing, I need to select No under Use Reference Annotation when I run Cufflink, and then use iGenome version of reference GTF when I run Cuffmerge? Yes, according to the tool documentation, this is the method. Based on your information and some protocols found online, my understanding is that: 1) if use iGenome version of reference GTF, I only need to run Cuffmerge with the Cufflink ouputs, because iGenome version reference GTF already contains attributes such as p_id and tss_id. Then the Cuffmerge output can be used for Cuffdiff. Yes, this is the example protocol I shared. 2) however, if I use the reference GTF from Ensembl/UCSC (rather than from iGenome), I need to run Cuffcompare to create p_id and tss_id, which is required for Cuffdiff. This can be tricky, it depends on what order you run the tools with and without the GTF annotation. The protocol in #1 is recommended. Am I right? Another question is: should I use iGenome version of reference GTF when I run Tophat if I want to see novel isoforms/splicing? Yes, this is what I intended to answer in my original reply, I apologize if that was not clear. The reference GTF can influence both mapping and assembly. So, both Tophat and Cufflinks. The information on the TopHat web site for the parameter provides more information (see link on TopHat tool form). The tool authors can also be contacted if there are some details that you are curious about that are not covered in the primary documentation: tophat.cuffli...@gmail.com Others are welcome to add to the thread with their experiences if they have used a reference annotation GTF with Tophat (or chosen not to for a particular reason that they would like to share), Best, Jen Galaxy team Thanks. Jianguang From: Jennifer Jackson [j...@bx.psu.edu] Sent: Thursday, August 23, 2012 11:46 AM To: Du, Jianguang Cc: galaxy-user@lists.bx.psu.edu Subject: Re: [galaxy-user] Should I use iGenomes verson of a reference GTF for Tophat? Hello Jianguang, When in the analysis process to start using the reference GTF file can depend on whether or not you intend to do any discovery along with differential expression testing. At the TopHat and Cufflinks steps, using reference GTF file can influence how datasets will map and assemble. In general, if your intention is to do discovery (e.g. work with novel isoforms in your data, but not in the reference), then do not add in the reference GTF until the CuffMerge step (to produce the input annotation GTF file for Cuffdiff). But if you want to guide the analysis toward known isoforms, then use the reference GTF. This is the process our RNA-seq example protocol follows: http://main.g2.bx.psu.edu/u/jeremy/p/galaxy-rna-seq-analysis-exercise For reference, there are other variations of this on the Cufflinks web site, some that never lead to Cuffdiff, but still may be useful to review. Please see the Cufflinks paper (linked from right side bar as Protocol for many more options/discussion. http://cufflinks.cbcb.umd.edu/tutorial.html -- Common uses of the Cufflinks package The end decision will be up to you, and a few runs with different options may be a useful way to make the final call, but hopefully this provides some resources to help you understand the option, Jen Galaxy team On 8/23/12 8:03 AM, Du, Jianguang wrote: Dear All, I am analysing RNA-seq datasets for differential splicing events between cell types. These are mouse cells. Jen suggested me to use the iGenomes version of reference GTF to take full advantage of the options in CuffDiff. My question is: should I use this iGenome version reference GTF when I run Tophat? Thanks. Jianguang ___ 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/ -- Jennifer Jackson http://galaxyproject.org -- Jennifer Jackson http://galaxyproject.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,
Re: [galaxy-user] Installing Galaxy and Hooking into a SGE Cluster
On Thu, Aug 23, 2012 at 8:44 PM, mailing list margeem...@gmail.com wrote: Anyway, back to my question. Does anyone know? Would I be better off asking on the developer mailing list or perhaps Stack Overflow? Software licensing is a much broader issue that Galaxy, so yes, perhaps Stack Overflow would be a better place. Peter ___ 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/
Re: [galaxy-user] Should I use iGenomes verson of a reference GTF for Tophat?
Hi Jen, I had a problem when I tried to run Tophat with the iGenome reference GTF. What I did is: 1) uploaded iGenome version of mm9 genes.gtf by: Shared Data - Data Libraries - iGenomes - click genes.gtf under mm9 - click Go for Import to current history. The genes.gtf appeared in history and turned green. 2) click Tophat for Illumina Find splice junctions using RNA-seq data to open the window of Tophat for Illumina (version 1.5.0) 3) selected the dataset to be analysed under RNA-Seq FASTQ file:. 4) chose Use one from the history under Will you select a reference genome from your history or use a built-in index?: Then the screen refreshed and the box (pulldown menu) under Select the reference genome: became smaller. Nothing showed up in the pulldown menu (actually the menu can not be pulled down). So that I could not input iGenome reference GTF. Looks like the Tophat can only Use a built-in index. How can I solve this problem? Thanks in advance. Jianguang From: galaxy-user-boun...@lists.bx.psu.edu [galaxy-user-boun...@lists.bx.psu.edu] on behalf of Du, Jianguang [jia...@iupui.edu] Sent: Thursday, August 23, 2012 4:01 PM To: Jennifer Jackson Cc: galaxy-user@lists.bx.psu.edu Subject: Re: [galaxy-user] Should I use iGenomes verson of a reference GTF for Tophat? Hi Jen, Thank you very much for your help. Jianguang From: Jennifer Jackson [j...@bx.psu.edu] Sent: Thursday, August 23, 2012 3:53 PM To: Du, Jianguang Cc: galaxy-user@lists.bx.psu.edu Subject: Re: [galaxy-user] Should I use iGenomes verson of a reference GTF for Tophat? Hello Jianguang, On 8/23/12 11:28 AM, Du, Jianguang wrote: Hi Jen, Thanks for your help. Do you mean that if I want to find novel isoform/splicing, I need to select No under Use Reference Annotation when I run Cufflink, and then use iGenome version of reference GTF when I run Cuffmerge? Yes, according to the tool documentation, this is the method. Based on your information and some protocols found online, my understanding is that: 1) if use iGenome version of reference GTF, I only need to run Cuffmerge with the Cufflink ouputs, because iGenome version reference GTF already contains attributes such as p_id and tss_id. Then the Cuffmerge output can be used for Cuffdiff. Yes, this is the example protocol I shared. 2) however, if I use the reference GTF from Ensembl/UCSC (rather than from iGenome), I need to run Cuffcompare to create p_id and tss_id, which is required for Cuffdiff. This can be tricky, it depends on what order you run the tools with and without the GTF annotation. The protocol in #1 is recommended. Am I right? Another question is: should I use iGenome version of reference GTF when I run Tophat if I want to see novel isoforms/splicing? Yes, this is what I intended to answer in my original reply, I apologize if that was not clear. The reference GTF can influence both mapping and assembly. So, both Tophat and Cufflinks. The information on the TopHat web site for the parameter provides more information (see link on TopHat tool form). The tool authors can also be contacted if there are some details that you are curious about that are not covered in the primary documentation: tophat.cuffli...@gmail.com Others are welcome to add to the thread with their experiences if they have used a reference annotation GTF with Tophat (or chosen not to for a particular reason that they would like to share), Best, Jen Galaxy team Thanks. Jianguang From: Jennifer Jackson [j...@bx.psu.edu] Sent: Thursday, August 23, 2012 11:46 AM To: Du, Jianguang Cc: galaxy-user@lists.bx.psu.edu Subject: Re: [galaxy-user] Should I use iGenomes verson of a reference GTF for Tophat? Hello Jianguang, When in the analysis process to start using the reference GTF file can depend on whether or not you intend to do any discovery along with differential expression testing. At the TopHat and Cufflinks steps, using reference GTF file can influence how datasets will map and assemble. In general, if your intention is to do discovery (e.g. work with novel isoforms in your data, but not in the reference), then do not add in the reference GTF until the CuffMerge step (to produce the input annotation GTF file for Cuffdiff). But if you want to guide the analysis toward known isoforms, then use the reference GTF. This is the process our RNA-seq example protocol follows: http://main.g2.bx.psu.edu/u/jeremy/p/galaxy-rna-seq-analysis-exercise For reference, there are other variations of this on the Cufflinks web site, some that never lead to Cuffdiff, but still may be useful to review. Please see the Cufflinks paper (linked from right side bar as Protocol for many more options/discussion. http://cufflinks.cbcb.umd.edu/tutorial.html -- Common uses of the Cufflinks package The end decision will be up to you, and a few runs with
Re: [galaxy-user] Installing galaxy with Apache ...
Hi Jelle, I'm still having issues with Apache. I've moved RewriteEngine on RewriteRule ^/galaxy$ /galaxy/ [R] RewriteRule ^/galaxy/static/style/(.*) /home/galaxy/galaxy-dist/static/june_2007_style/blue/$1 [L] RewriteRule ^/galaxy/static/scripts/(.*) /home/galaxy/galaxy-dist/static/scripts/packed/$1 [L] RewriteRule ^/galaxy/static/(.*) /home/galaxy/galaxy-dist/static/$1 [L] RewriteRule ^/galaxy/favicon.ico /home/galaxy/galaxy-dist/static/favicon.ico [L] RewriteRule ^/galaxy/robots.txt /home/galaxy/galaxy-dist/static/robots.txt [L] RewriteRule ^/galaxy(.*) http://localhost:8080$1 [P] from /var/www/.htaccess to /etc/apache2/httpd.conf I still get a 404 Not found error when trying to access galaxy (http://140.253.78.44/galaxy ) http://140.253.78.44 still gets the It Works! Apache default index.html and http://140.253.78.44:8080 gets the galaxy page /var/log/apache2/error.log states... [Fri Aug 24 07:35:27 2012] [error] [client 140.253.78.44] File does not exist: /var/www/favicon.ico [Fri Aug 24 07:36:25 2012] [error] [client 140.253.78.44] File does not exist: /var/www/galaxy Now if I change httpd.conf and add the vitrtualHost tags to: ServerName localhost VirtualHost *:80 RewriteEngine on RewriteRule ^/galaxy$ /galaxy/ [R] RewriteRule ^/galaxy/static/style/(.*) /home/galaxy/galaxy-dist/static/june_2007_style/blue/$1 [L] RewriteRule ^/galaxy/static/scripts/(.*) /home/galaxy/galaxy-dist/static/scripts/packed/$1 [L] RewriteRule ^/galaxy/static/(.*) /home/galaxy/galaxy-dist/static/$1 [L] RewriteRule ^/galaxy/favicon.ico /home/galaxy/galaxy-dist/static/favicon.ico [L] RewriteRule ^/galaxy/robots.txt /home/galaxy/galaxy-dist/static/robots.txt [L] RewriteRule ^/galaxy(.*) http://localhost:8080$1 [P] /VirtualHost I now get a 403 Forbidden error when trying to access (http://140.253.78.44/galaxy ) You don't have permission to access /galaxy/ on this server, and /var/log/apache2/error.log states.. [Fri Aug 24 07:45:33 2012] [error] [client 140.253.78.44] attempt to make remote request from mod_rewrite without proxy enabled: proxy:http://localhost:8080/ [Fri Aug 24 07:45:34 2012] [error] [client 140.253.78.44] File does not exist: /etc/apache2/htdocs Any help much appreciated Neil From: Jelle Scholtalbers [j.scholtalb...@gmail.com] Sent: Thursday, August 23, 2012 8:20 PM To: Burdett, Neil (ICT Centre, Herston - RBWH) Cc: galaxy-user@lists.bx.psu.edu Subject: Re: [galaxy-user] Installing galaxy with Apache ... Hi Neil, with your current apache configuration, you should probably see Galaxy at http://yourip/galaxy This is due to your Rewriterule /galaxy and for that you have also set proxy_prefix = /galaxy in your universe_wsgi.ini You should probably remove the empty galaxy directory under /var/www Furthermore, most of the time you will not need a directory with 777 in your apache web/documentroot.. Cheers, Jelle On Thu, Aug 23, 2012 at 8:34 AM, neil.burd...@csiro.au wrote: Hi I've installed galaxy with the default settings and it works fine. On a new Ubuntu machine I am trying to get galaxy running with Apache. But I am having problems, I've followed the documentation. After installing Apache I can put my ip address into a browser and I get message saying Apache is working fine. It displays a message from /var/www/index.html Now when I start galaxy I was assuming I would get redirected to the galaxy welcome page, but this doesn't happen and it remains at /var/www/index.html. To view the welcome page I have to add :8080 after the ip address. Is this still required? I thought that Apache would know to redirect to my distributionWhat am I doing wrong? /etc/apache2/sites-available/default-ssl and /etc/apache2/sites-available/default look like this ... DocumentRoot /var/www Directory / Options FollowSymLinks AllowOverride None /Directory Directory /var/www/ Options Indexes FollowSymLinks MultiViews AllowOverride None Order allow,deny allow from all /Directory etc ... /var/www/.htaccess RewriteEngine on RewriteRule ^/galaxy$ /galaxy/ [R] RewriteRule ^/galaxy/static/style/(.*) /home/galaxy/galaxy-dist/static/june_2007_style/blue/$1 [L] RewriteRule ^/galaxy/static/scripts/(.*) /home/galaxy/galaxy-dist/static/scripts/packed/$1 [L] RewriteRule ^/galaxy/static/(.*) /home/galaxy/galaxy-dist/static/$1 [L] RewriteRule ^/galaxy/favicon.ico /home/galaxy/galaxy-dist/static/favicon.ico [L] RewriteRule ^/galaxy/robots.txt /home/galaxy/galaxy-dist/static/robots.txt [L] RewriteRule ^/galaxy(.*) http://localhost:8080$1 [P] I created an empty 'galaxy' directory with 777 permissions on it under /var/www and universe_wsgi.ini # Define the proxy-prefix filter. [filter:proxy-prefix] use = egg:PasteDeploy#prefix prefix = /galaxy #
Re: [galaxy-user] Should I use iGenomes verson of a reference GTF for Tophat?
Hello Jianguang, Two different data are being mixed up: genome vs annotation reference genome (format: fasta) vs reference annotation (format: GTF) To annotation your sequences against the mm9 reference genome, choose locally cashed and select mm9 from the pull down menu. Then, optionally, if you want to guide the mapping with a reference annotation GTF file, that is what the genes.gtf file represents. The option is set on the TopHat form under: TopHat settings to use: Full Paramater list Use Own Junctions: Yes Use Gene Annotation Model: Yes Gene Model Annotations: select the dataset with the GTF file Best, Jen Galaxy team On 8/23/12 2:48 PM, Du, Jianguang wrote: Hi Jen, I had a problem when I tried to run Tophat with the iGenome reference GTF. What I did is: 1) uploaded iGenome version of mm9 genes.gtf by: Shared Data - Data Libraries - iGenomes - click genes.gtf under mm9 - click Go for Import to current history. The genes.gtf appeared in history and turned green. 2) click Tophat for Illumina Find splice junctions using RNA-seq data to open the window of Tophat for Illumina (version 1.5.0) 3) selected the dataset to be analysed under RNA-Seq FASTQ file:. 4) chose Use one from the history under Will you select a reference genome from your history or use a built-in index?: Then the screen refreshed and the box (pulldown menu) under Select the reference genome: became smaller. Nothing showed up in the pulldown menu (actually the menu can not be pulled down). So that I could not input iGenome reference GTF. Looks like the Tophat can only Use a built-in index. How can I solve this problem? Thanks in advance. Jianguang From: galaxy-user-boun...@lists.bx.psu.edu [galaxy-user-boun...@lists.bx.psu.edu] on behalf of Du, Jianguang [jia...@iupui.edu] Sent: Thursday, August 23, 2012 4:01 PM To: Jennifer Jackson Cc: galaxy-user@lists.bx.psu.edu Subject: Re: [galaxy-user] Should I use iGenomes verson of a reference GTF for Tophat? Hi Jen, Thank you very much for your help. Jianguang From: Jennifer Jackson [j...@bx.psu.edu] Sent: Thursday, August 23, 2012 3:53 PM To: Du, Jianguang Cc: galaxy-user@lists.bx.psu.edu Subject: Re: [galaxy-user] Should I use iGenomes verson of a reference GTF for Tophat? Hello Jianguang, On 8/23/12 11:28 AM, Du, Jianguang wrote: Hi Jen, Thanks for your help. Do you mean that if I want to find novel isoform/splicing, I need to select No under Use Reference Annotation when I run Cufflink, and then use iGenome version of reference GTF when I run Cuffmerge? Yes, according to the tool documentation, this is the method. Based on your information and some protocols found online, my understanding is that: 1) if use iGenome version of reference GTF, I only need to run Cuffmerge with the Cufflink ouputs, because iGenome version reference GTF already contains attributes such as p_id and tss_id. Then the Cuffmerge output can be used for Cuffdiff. Yes, this is the example protocol I shared. 2) however, if I use the reference GTF from Ensembl/UCSC (rather than from iGenome), I need to run Cuffcompare to create p_id and tss_id, which is required for Cuffdiff. This can be tricky, it depends on what order you run the tools with and without the GTF annotation. The protocol in #1 is recommended. Am I right? Another question is: should I use iGenome version of reference GTF when I run Tophat if I want to see novel isoforms/splicing? Yes, this is what I intended to answer in my original reply, I apologize if that was not clear. The reference GTF can influence both mapping and assembly. So, both Tophat and Cufflinks. The information on the TopHat web site for the parameter provides more information (see link on TopHat tool form). The tool authors can also be contacted if there are some details that you are curious about that are not covered in the primary documentation: tophat.cuffli...@gmail.com Others are welcome to add to the thread with their experiences if they have used a reference annotation GTF with Tophat (or chosen not to for a particular reason that they would like to share), Best, Jen Galaxy team Thanks. Jianguang From: Jennifer Jackson [j...@bx.psu.edu] Sent: Thursday, August 23, 2012 11:46 AM To: Du, Jianguang Cc: galaxy-user@lists.bx.psu.edu Subject: Re: [galaxy-user] Should I use iGenomes verson of a reference GTF for Tophat? Hello Jianguang, When in the analysis process to start using the reference GTF file can depend on whether or not you intend to do any discovery along with differential expression testing. At the TopHat and Cufflinks steps, using reference GTF file can influence how datasets will map and assemble. In general, if your intention is to do discovery (e.g. work with novel isoforms in your data, but not in the reference), then do not add in the reference GTF until the CuffMerge step (to produce the
