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Is tss_id unique for each transcript?
by Du, Jianguang 24 Aug '12

24 Aug '12

Dear All, I am reading and comparing the outputs of Cuffdiff. I found there is a "tss_id" column in "transcript FPKM tracking", "gene FPKM tracking", "TSS groups FPKM tracking" and "CDS FPKM tracking" files. In this column, each line has a unique label such as "TSS1001". This kind of labels also appear under "test_id" column in "TSS groups differential expression testing" and "splicing defferential expression testing" files. Does each of this kind label represent a unique transcript? In other word, does each transcript has a unique label initiating with TSS followed by numbers? Thank. Jianguang

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regarding upload of data locally on galaxy
by Amit Pande 24 Aug '12

24 Aug '12

Dear galaxy, I have a local instance of Galaxy running on the server and my problem is as follows- I want to analyze chip seq data from ENCODE project using the NGS tools but since the BAM files are too big to be uploaded, I am having difficulty in my work. Could you be kind enough to point out as to how to upload the files vis ftp as mentioned ??? is there a tutorial available ? warm regards, Amit.

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Re: [galaxy-user] Should I use iGenomes verson of a reference GTF for Tophat?
by Du, Jianguang 24 Aug '12

24 Aug '12

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-bounces(a)lists.bx.psu.edu [galaxy-user-bounces(a)lists.bx.psu.edu] on behalf of Du, Jianguang [jiandu(a)iupui.edu] Sent: Thursday, August 23, 2012 4:01 PM To: Jennifer Jackson Cc: galaxy-user(a)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 [jen(a)bx.psu.edu] Sent: Thursday, August 23, 2012 3:53 PM To: Du, Jianguang Cc: galaxy-user(a)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.cufflinks(a)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 [jen(a)bx.psu.edu] > Sent: Thursday, August 23, 2012 11:46 AM > To: Du, Jianguang > Cc: galaxy-user(a)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, please use the interface at: http://lists.bx.psu.edu/

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Installing Galaxy and Hooking into a SGE Cluster
by mailing list 23 Aug '12

23 Aug '12

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

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Should I use iGenomes verson of a reference GTF for Tophat?
by Du, Jianguang 23 Aug '12

23 Aug '12

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

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How much can I trimm my reads
by Du, Jianguang 23 Aug '12

23 Aug '12

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

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tophat deletions output
by i b 23 Aug '12

23 Aug '12

dear all, how can we use the tophat deletions output? e.g. if I want to see and conpare between two samples if a specific gene or transcript had been deleted, how can I use this output? is visualisation enough? thanks, ib

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inconsistent cuffdiff output
by i b 23 Aug '12

23 Aug '12

Dear all, I have two samples, treated and untreated. I ran cufflinks and had the following: treated: 884532 untreat.: 130247 I then ran cuffdiff and look ath transcript diff. expression: treated: 33 untreat.:2.9 significant:yes Strangely for another transcripts I have: Cufflinks: treat_:0 untreat_: 0 Cuffdiff: trat_4.4 untreat_0.2 significant: yes How this can be possible? I had similar results with other transcripts where one one of the two had 0 cufflinks, and the other one very high but the cuffdiff come out with NO TEST or OK but not significant thanks, ib

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How to find the alternatively spliced segment of genes in Cuffdiff output
by Du, Jianguang 23 Aug '12

23 Aug '12

Dear All, I have run programs from Tophat to Cuffdiff of Galaxy to look for the difference in alternative splicing events between cell types. However I do not know how to find the detail information (such as the sequence and the genomic coordinates) of the alternatively spliced part of a given gene. I looked at the data of Cuffdiff ouput " splicing differential expression testing", there is no column showing the position of alternatively spliced region. Please help to solve this problem. Thanks in advance. Jianguang Du

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How to upload multiple files......
by Neil.Burdett＠csiro.au 23 Aug '12

23 Aug '12

Hi, I've recently installed galaxy. I was trying to find how to upload multiple files without the need to compress them into a zip/gz file first. Is this possible? As the "Get Data" option allows only one file at a time. I searched the galaxy archive mailing list but didn't find anything conclusive. I am unable to use ftp client tools due to IT restrictions. Thanks Neil

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