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Re: [galaxy-user] cufflinks FPKM
by Jeremy Goecks 31 Mar '11

31 Mar '11

If Cufflinks works without a reference GTF, then the problem is the mismatch b/t your GTF and your bam file. A couple things to check: (1) that your genome chrom names in F3.csfata match those in your GTF; if not, you'll need to modify your GTF to match the names in your fasta. (2) that your GTF is sorted as your BAM is sorted. If these issues don't solve your problem, it's best to use the Cufflinks help email that I noted in my previous email. Good luck, J. On Mar 31, 2011, at 10:32 AM, lishiyong wrote: > > Hello,I don't use Galaxy.But I have uploaded the file(sorted file 20:test_44.bam refgene.gtf : ) It's works OK without the reference GTF file ,while with reference, I can't gain the right results, I do these in my computer. > 2011-03-31 > lishiyong > 发件人： Jeremy Goecks > 发送时间： 2011-03-31 21:36:52 > 收件人： lishiyong > 抄送： galaxy-user > 主题： Re: [galaxy-user] cufflinks FPKM > Hello, > > It's not clear whether you're using Galaxy. If you're using Galaxy, please share you history with me (History Options --> Share/Publish --> Share with User --> my email) and I'll take a look; otherwise, Cufflinks has an email list for questions: tophat.cufflinks(a)gmail.com > > Best, > J. > > On Mar 31, 2011, at 3:39 AM, lishiyong wrote: > >> Hi: >> >> >> I gain the SOLiD sequencing data.I used bowtie to map human genome then I sort the sam file .I used cuffinks to calculate FPKM with the sam file ,human gtf file .it gives 0 FPKM values and this is for all genes .what's the reason? >> >> (1) bowtie -C human_hg18_color -f F3.csfasta -Q F3_QV.qual -v 2 -k 100 -p 6 --mapq --sam test.sam >> (2) samtools view -uS test.sam 2>/dev/null | samtools sort -m 2000000000 - test.bam >> (3) cufflinks -G refGene_hg18.gtf test.bam.bam >> 2011-03-31 >> lishiyong >> ___________________________________________________________ >> 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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cufflinks FPKM
by lishiyong 31 Mar '11

31 Mar '11

Hi: I gain the SOLiD sequencing data.I used bowtie to map human genome then I sort the sam file .I used cuffinks to calculate FPKM with the sam file ,human gtf file .it gives 0 FPKM values and this is for all genes .what's the reason? (1) bowtie -C human_hg18_color -f F3.csfasta -Q F3_QV.qual -v 2 -k 100 -p 6 --mapq --sam test.sam (2) samtools view -uS test.sam 2>/dev/null | samtools sort -m 2000000000 - test.bam (3) cufflinks -G refGene_hg18.gtf test.bam.bam 2011-03-31 lishiyong

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NR data for instantiation
by drhoads＠uark.edu 31 Mar '11

31 Mar '11

My IT people are setting us up for an instantiation of Galaxy on campus for my course and faculty to use. They have most everything working on our server but when I get to the Megablast there are no databases. I need to give them the instructions for how to set up for databases like NR and maybe the human HGTS. Where can I find that information to give to the people that will understand it? Douglas Rhoads, Professor of Biological Sciences Director of Graduate Program in Cell and Molecular Biology 601 SCEN, Dept. of Biological Sciences, 1 University of Arkansas, Fayetteville, AR 72701 drhoads(at)uark.edu, http://biscweb.uark.edu/drhoads phone: 479-575-3251 FAX: 479-575-4010

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aaChanges / BED-to-Codon
by Christopher Schroeder 31 Mar '11

31 Mar '11

Hello list, I already analysed some of our data with your framework and I want to thank you for your great work. But lately I stumbled on some strange behavior - I used the "aaChanges"-tool, as well as the Gene "BED-to-Codon"-tool with the same Gene-BED-File and compared the results. What I did: #example BED-file: chr17 3763624 3794037 uc002fwt.2 0 - 3765504 3788981 0 16 1953,104,129,88,74,54,200,89,22,37,134,52,54,48,403,51, 0,5577,9156,9470,12223,13078,15892,20015,21296,22197,22711,23098,23553,24049 ,24997,30362, #example-output of "aaChanges" chr17 3775847 3775848 C uc002fwt.2 Glu:Gly 375 T #example-output of "BED-to-Codon" chr17 3773172 3773175 uc002fwt.2 0 - chr17 3773175 3773178 uc002fwt.2 0 - chr17 3773178 3773181 uc002fwt.2 0 - chr17 3775849 3775852 uc002fwt.2 0 - chr17 3775852 3775855 uc002fwt.2 0 - Comparing both results, the "BED-to-Codon" actually presents no codon at position 3775848 - here the fist codon is starting at 3775849. But in fact, looking at the ucsc browser, "aaChanges" is right. There are another few examples (~5/1500 snps) and apparently most of them are positions among the first codon in an exon. Did anyone meet this problem before? What is my mistake? Thank you. Regards Chris

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labeling sections of the history
by Ryan Golhar 30 Mar '11

30 Mar '11

I'm analyzing a bunch of datasets in a single history and the number of history items is getting a little confusing. Is it possible to attached a label a group of history items, or collapse a group of history items on the display? I don't suppose it is, but it would be a nice feature... -- CONFIDENTIALITY NOTICE: This email communication may contain private, confidential, or legally privileged information intended for the sole use of the designated and/or duly authorized recipient(s). If you are not the intended recipient or have received this email in error, please notify the sender immediately by email and permanently delete all copies of this email including all attachments without reading them. If you are the intended recipient, secure the contents in a manner that conforms to all applicable state and/or federal requirements related to privacy and confidentiality of such information.

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Re: [galaxy-user] Trouble with RNAseq analysis
by Jeremy Goecks 30 Mar '11

30 Mar '11

Cristian, The ">" is a formatting character; what needs to match is the string after the ">" in the genome file and the entries in the contig column of your GTF. Your GTF is quite different that your genome file; your genome file has 10 contigs labeled by number, but your GTF has many, many contig names labelled by numbers and names. For Cufflinks to work, you can either (a) turn off bias correction or (b) restrict entries in your GTF to those that match your reference genome. Finally, please reply all to emails so that all emails remain on list for archival and community purposes. Thanks, J. On Mar 30, 2011, at 12:02 PM, Cristian Rojas wrote: > Thanks Jeremy. But in genomes fasta files very often any chromosome represents a > sequence followed by ">". Then, it is no possible match contig names in GTF with > ">" names in Genome fasta. What must I do? > Cristian > > > > ----- Mensaje original ---- > De: Jeremy Goecks <jeremy.goecks(a)emory.edu> > Para: Cristian Rojas <cristianrojasbr(a)yahoo.com.ar> > CC: galaxy-user(a)lists.bx.psu.edu > Enviado: miércoles, 30 de marzo, 2011 12:53:50 > Asunto: Re: [galaxy-user] Trouble with RNAseq analysis > > Cristian, > > The contig names in your GTF file don't match those in your reference (fasta) > file. In order for Cufflinks to use a reference GTF, its contigs names must > match those in your reference genome. > > Best, > J. > > On Mar 30, 2011, at 11:31 AM, Cristian Rojas wrote: > >> Thanks Jeremy. I did it. >> Cristian >> >> >> >> ----- Mensaje original ---- >> De: Jeremy Goecks <jeremy.goecks(a)emory.edu> >> Para: Cristian Rojas <cristianrojasbr(a)yahoo.com.ar> >> CC: galaxy-user(a)lists.bx.psu.edu >> Enviado: miércoles, 30 de marzo, 2011 12:02:47 >> Asunto: Re: [galaxy-user] Trouble with RNAseq analysis >> >> Cristian, >> >> Please share your history with me (History Options --> Share/Publish --> Share > >> with User --> my email) and I'll take a look. >> >> Thanks, >> J. >> >> On Mar 30, 2011, at 10:48 AM, Cristian Rojas wrote: >> >>> Hi everybody, I am trying to analyze the differential expression between two >>> RNAseq samples. But I found many troubles aligning my reads. I will describe >>> what I did. First I groomed the FastQ files (2). Then I uploaded the Sorghum >>> genome and aligned the reads to it with Tophat. Aftter that, I tried to use >>> Cufflink with the BAM file of Tophat, using as annotation file an uploaded GTF >> >> >>> file and the Sorghum genome, but I received an error message in the three >>> outputs of Cufflink. I tried to align against new brand Maize genome (now at >>> Galaxy), and the same messages. I also converted the BAM file to SAM, but the > >>> same. Any advice? What was wrong? >>> Thanks in advance. >>> Cristian >>> >>> >>> ___________________________________________________________ >>> 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: [galaxy-user] Trouble with RNAseq analysis
by Jeremy Goecks 30 Mar '11

30 Mar '11

Cristian, The contig names in your GTF file don't match those in your reference (fasta) file. In order for Cufflinks to use a reference GTF, its contigs names must match those in your reference genome. Best, J. On Mar 30, 2011, at 11:31 AM, Cristian Rojas wrote: > Thanks Jeremy. I did it. > Cristian > > > > ----- Mensaje original ---- > De: Jeremy Goecks <jeremy.goecks(a)emory.edu> > Para: Cristian Rojas <cristianrojasbr(a)yahoo.com.ar> > CC: galaxy-user(a)lists.bx.psu.edu > Enviado: miércoles, 30 de marzo, 2011 12:02:47 > Asunto: Re: [galaxy-user] Trouble with RNAseq analysis > > Cristian, > > Please share your history with me (History Options --> Share/Publish --> Share > with User --> my email) and I'll take a look. > > Thanks, > J. > > On Mar 30, 2011, at 10:48 AM, Cristian Rojas wrote: > >> Hi everybody, I am trying to analyze the differential expression between two >> RNAseq samples. But I found many troubles aligning my reads. I will describe >> what I did. First I groomed the FastQ files (2). Then I uploaded the Sorghum >> genome and aligned the reads to it with Tophat. Aftter that, I tried to use >> Cufflink with the BAM file of Tophat, using as annotation file an uploaded GTF > >> file and the Sorghum genome, but I received an error message in the three >> outputs of Cufflink. I tried to align against new brand Maize genome (now at >> Galaxy), and the same messages. I also converted the BAM file to SAM, but the >> same. Any advice? What was wrong? >> Thanks in advance. >> Cristian >> >> >> ___________________________________________________________ >> 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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Trouble with RNAseq analysis
by Cristian Rojas 30 Mar '11

30 Mar '11

Hi everybody, I am trying to analyze the differential expression between two RNAseq samples. But I found many troubles aligning my reads. I will describe what I did. First I groomed the FastQ files (2). Then I uploaded the Sorghum genome and aligned the reads to it with Tophat. Aftter that, I tried to use Cufflink with the BAM file of Tophat, using as annotation file an uploaded GTF file and the Sorghum genome, but I received an error message in the three outputs of Cufflink. I tried to align against new brand Maize genome (now at Galaxy), and the same messages. I also converted the BAM file to SAM, but the same. Any advice? What was wrong? Thanks in advance. Cristian

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{Disarmed} extra hack to get own database connection to work?
by Perdacher,Martin 30 Mar '11

30 Mar '11

hello galaxy-community, we stored a lot of peak-information in a database. to import these files into galaxy, i read the DataConnectionProtocol (https://bitbucket.org/galaxy/galaxy-central/wiki/DataSources) and i also took a look at the biomart.xml example (https://bitbucket.org/galaxy/galaxy-dist/src/8729d2e29b02/tools/data_source…) biomart does exactly what i want. here is my full tool configuration xml-file: <tool id="regionDb" name="RegionDb-Import"> <description>Import from regionDb</description> <command interpreter="python">../data_source/data_source.py $output $__app__.config.output_size_limit</command> <inputs action="http://localhost:8888/index.html" check_values="false" method="get"> <display>Display-Text</display> <param name="GALAXY_URL" type="baseurl" value="/tool_runner" /> <param name="tool_id" type="hidden" value="regionDb" /> </inputs> <request_param_translation> <request_param galaxy_name="URL" remote_name="URL" missing=""/> <request_param galaxy_name="URL_method" remote_name="URL_method" missing="get" /> <request_param galaxy_name="name" remote_name="name" missing="Biomart test query" /> </request_param_translation> <outputs> <data name="regionDb File" format="bed" label="regionDb import" /> </outputs> <help> **What it does** import ... </help> </tool> content of http://localhost:8888/index.html: <form method="POST" type="text" action="http://galaxy/tool_runner/regionDb?type=text&name=new%20history%20entry&URL…" > <input type="submit" value="Send to Galaxy"/> </form> i also tried a different action="http://galaxy/tool_runner?tool_id=regionDb&type=text&name=new%20history%20e…" with the same error message (shown below). what actually works is, if i try to receive biomart data with this request. <form method="POST" type="text" action="http://gen43/galaxy/tool_runner/biomart?type=text&name=ne%20history%20entry…" > <input type="submit" value="Send to Galaxy"/> </form> my question is: was there some extra hack to get the biomart tool to run? i found some lines in the lib/galaxy/web/controllers/tool_runner.py which confirm that assumption: #Hack to get biomart to work, ideally, we could pass tool_id to biomart and receive it back @web.expose def biomart(self, trans, tool_id='biomart', **kwd): """Catches the tool id and redirects as needed""" return self.index(trans, tool_id=tool_id, **kwd) Server Error URL: http://galaxy/tool_runner/regionDb?type=text&name=new%20history%20entry&URL… Module paste.exceptions.errormiddleware:143 in __call__ >> app_iter = self.application(environ, start_response) Module paste.debug.prints:98 in __call__ >> environ, self.app) Module paste.wsgilib:539 in intercept_output >> app_iter = application(environ, replacement_start_response) Module paste.recursive:80 in __call__ >> return self.application(environ, start_response) Module galaxy.web.framework.middleware.remoteuser:109 in __call__ >> return self.app( environ, start_response ) Module paste.httpexceptions:632 in __call__ >> return self.application(environ, start_response) Module galaxy.web.framework.base:145 in __call__ >> body = method( trans, **kwargs ) Module galaxy.web.controllers.tool_runner:27 in regionDb >> return self.index(trans, tool_id=tool_id, **kwd) Module galaxy.web.controllers.tool_runner:75 in index >> template, vars = tool.handle_input( trans, params.__dict__ ) Module galaxy.tools:933 in handle_input >> _, out_data = self.execute( trans, incoming=params ) Module galaxy.tools:1225 in execute >> return self.tool_action.execute( self, trans, incoming=incoming, set_output_hid=set_output_hid, history=history ) Module galaxy.tools.actions:291 in execute >> wrap_values( tool.inputs, params ) Module galaxy.tools.actions:154 in wrap_values >> input_values[ input.name ] = galaxy.tools.InputValueWrapper( input, input_values[ input.name ], incoming ) KeyError: 'GALAXY_URL' extra data CGI Variables CONTENT_LENGTH '-1' CONTENT_TYPE 'application/x-www-form-urlencoded' HTTP_ACCEPT 'text/html,application/xhtml+xml,application/xml;q=0.9,*/*;q=0.8' HTTP_ACCEPT_CHARSET 'ISO-8859-1,utf-8;q=0.7,*;q=0.7' HTTP_ACCEPT_ENCODING 'gzip,deflate' HTTP_ACCEPT_LANGUAGE 'en-us,en;q=0.5' HTTP_CONNECTION 'Keep-Alive' HTTP_COOKIE 'galaxysession=c6ca0ddb55be603af8ffade046cb8ca7fe7a18f326db29b947c846a3b24831e1362e15c491e90b43' HTTP_HOST 'gen43' HTTP_REFERER 'http://localhost:8888/index2.php?GALAXY_URL=http%3A//gen43/galaxy/tool_runn…' HTTP_REMOTE_USER 'perdacher(a)genau.at' HTTP_USER_AGENT 'Mozilla/5.0 (Macintosh; U; Intel Mac OS X 10.6; en-US; rv:1.9.2.3) Gecko/20100401 Firefox/3.6.3' HTTP_VIA '1.1 gen43.imp.univie.ac.at' PATH_INFO '/tool_runner/regionDb' QUERY_STRING 'type=text&name=new%20history%20entry&URL=http://localhost:8888/getfile.php' REMOTE_ADDR '10.42.111.27' REQUEST_METHOD 'POST' SCRIPT_NAME '/galaxy' SERVER_NAME 'gen43.imp.univie.ac.at' SERVER_PORT '9191' SERVER_PROTOCOL 'HTTP/1.1' Configuration __file__ '/projects/solexadst/serverData/galaxy-devel/universe_wsgi.webapp.ini' admin_users 'tamir(a)genau.at' apache_xsendfile 'False' brand 'GenAu-Epx Development!!!' cookie_path '/galaxy' debug 'TRUE' enable_api 'True' enable_job_running 'False' enable_pages 'True' here '/projects/solexadst/serverData/galaxy-devel' log_level 'DEBUG' new_user_dataset_access_role_default_private 'True' remote_user_logout_href '/logout' remote_user_maildomain 'genau.at' require_login 'True' set_metadata_externally 'True' static_cache_time '360' static_dir '/projects/solexadst/serverData/galaxy-devel/static/' static_enabled 'True' static_favicon_dir '/projects/solexadst/serverData/galaxy-devel/static/favicon.ico' static_images_dir '/projects/solexadst/serverData/galaxy-devel/static/images' static_scripts_dir '/projects/solexadst/serverData/galaxy-devel/static/scripts/' static_style_dir '/projects/solexadst/serverData/galaxy-devel/static/june_2007_style/blue' upstream_gzip 'False' use_interactive 'False' use_remote_user 'True'

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Combining the paired reads from Illumina run
by Surya Saha 30 Mar '11

30 Mar '11

Hi, I have two fastq files with the forward(/1) and reverse(/2) paired reads. The reads are not in same order in either file, some pairs are absent/missing and the files are 8 GB each with abt 30 mill reads each. I am trying to pull out all the paired reads for which both fwd and rev exist. Can I use a combination of fastq tools in Galaxy to do this? Thanks! -Surya

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