Re: [galaxy-user] Using galaxy for Bacterial RNA-seq
Dear Noa and Bomba i am also in situation like you, hardly any programming experiance but want to analyse, bacterial tranmscriptome before and after an stress. I am very new to galaxy, would be obliged if any one can give me more hints, i have few queries In Brief; we did our sequnecing by Hiseq 2000, from our partner i got two files (FASTQ) for each treatment, for example R1 and R2 i was sucessful to upload these files (Fastq) and reference genome file (Fasta) via FTP, after upload, i run the Fastq groomer, with my understanding i must quality filter my sequences, but i am not sure what shoud be the cutoff. after reading Noa email, seems like i should do Bowtie mapping, after groomer , and filter is is there any other middle step also before i go to bowtie Another question is, should i join both the files (fastq joiner) at any stage of analysis Bomba could you please send me link as you stated, for RNA seqq analysis on Univerity of albama website, although it is for eukaryote, but may be will help me to understand the steps Thanking you all From: Noa Sher noa.s...@gmail.com To: Jeremy Goecks jeremy.goe...@emory.edu Cc: galaxy-user@lists.bx.psu.edu (galaxy-user@lists.bx.psu.edu) galaxy-user@lists.bx.psu.edu; closetic...@galaxyproject.org; Bomba Dam bomba@mpi-marburg.mpg.de Sent: Thursday, February 16, 2012 8:31 PM Subject: Re: [galaxy-user] Using galaxy for Bacterial RNA-seq Hi Bomba I have been recently struggling with the same issue (RNA-Seq on a bacteria; no programming experience). I was in touch with the authors of the tophat-cufflinks suite and they all suggested that given that bacteria have little or no introns, you can do bowtie followed by cufflinks and skip the tophat. Alternatively, if you do decide to do tophat and then cufflinks, make SURE to change the intron size parameters, otherwise you will get a mess. I used min intron distance 10bp, max size 1000bp. If you just want to do comparative work you can do bowtie and then cuffdiff directly. Look at your data with the Galaxy genome browser after you run tophat or bowtie to make sure it looks OK. (I found it easier to convert bowtie data from SAM to BAM file and then to view it). One thing I havent fully looked into is what happens at the ends of the mapping (since bowtie assumes linear chromosomes and not circularized, so just be aware of that difference as not all reads will align properly at the ends). Feel free to contact me if you need more help - I am definitely not an expert but have been struggling through doing RNA-Seq on galaxy for the past month or so, so may be able to help with some things. Make sure you use matching gtf reference annotation (if you have this) and genome! Good luck! noa On 16/02/2012 20:01, Jeremy Goecks wrote: Bomba, I'm not familiar enough with bacterial/prokaryotic transcriptomes to suggest a possible workflow. You might try the standard Tophat-Cufflinks-Cuffcompare/merge-Cuffdiff workflow and see whether you get meaningful results; Tophat runs Bowtie internally, so there's no reason to run Bowtie separately unless there are Bowtie-specific parameters that you need to modify. I've had very little experience with PALMapper and can't speak to its efficacy, either for eukaryotic or prokaryotic transcriptome analyses. Finally, I've cc'd the galaxy-user mailing list. Using this list is the best way to reach the Galaxy user community and get in touch with someone that has used Galaxy to analyze bacterial transcriptomes. Good luck, J. On Feb 16, 2012, at 9:17 AM, Bomba Dam wrote: Dear Dr. Goecks, I am working as a post-doctoral fellow in MPI Marburg, Germany. We am trying to understand the differential expression of genes in a methanotrophic bacterium under different growth conditions. We are sequencing the transcriptome using Illumina Hiseq. As I dont have expertise in programming languages, I found the Galaxy interface very user-friendly for doing such transcriptome analysis. However, I could not find a step wise protocol\workflow for mapping bacterial RNA-seq against the reference genome (we have the completely sequenced genome of our model organism). I have found a detailed step by step workflow for RNA-seq analysis from the University of Alabama web-site (uab.edu). However, it refers to the eukaryotic system. Most examples provided and used for analysis are from eukaryotic systems. I am a bit confused weather the same workflow will also work well for bacterial systems as there are no splicing events or I should make some modifications. Can you kindly suggest me which workflow should I follow for mapping the bacterial reads (Bowtie, Tophat or PALMapper) and subsequent quantification steps. I want some guidance in this regard. With kind regards, Bomba Dam -- Dr. BOMBA DAM Alexander von Humboldt Postdoctoral Research Fellow Max-Planck-Institut für terrestrische Mikrobiologie
Re: [galaxy-user] Error on custom builds on public galaxy server
Yeah, Hope we could find the answer from Galaxy team soon :-) Pengfei On Thu, Feb 16, 2012 at 11:00 PM, Noa Sher noa.s...@gmail.com wrote: I also had exactly the same problem yesterday - glad to see that maybe it wasn't just me. Happy for help! Noa On 16/02/2012 22:24, Pengfei Yu wrote: Dear sir or madam, I am using galaxy public server and I am trying to build a custom genome using the function custom Builds in user section. I specified the Name, Key and uploaded the genome fasta file also the chromosome length file. But when I click submit, galaxy gave an error information like that: *Server Error:* An error occurred. See the error logs for more information. (Turn debug on to display exception reports here) I don't know where to find the error logs and how to turn debug on. Could you tell me how to fix the problem and upload the custom builds? Thanks a lot! Pengfei -- Pengfei Yu Center of Biophysics and Computational Biology University of Illinois Urbana-Champaign Illinois, 61820, US Cell phone: 217-898-6301 ___ 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/ -- Pengfei Yu Center of Biophysics and Computational Biology University of Illinois Urbana-Champaign Illinois, 61820, US Cell phone: 217-898-6301 ___ 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] Error on custom builds on public galaxy server
Hello Pengfei, Noa, Galaxy was experiencing intermittent cluster failures yesterday. Please run your jobs again. I was able to create a test build a few minutes ago without a problem at the public Main instance. We are very sorry for the inconvenience, Jen Galaxy team On 2/16/12 12:24 PM, Pengfei Yu wrote: Dear sir or madam, I am using galaxy public server and I am trying to build a custom genome using the function custom Builds in user section. I specified the Name, Key and uploaded the genome fasta file also the chromosome length file. But when I click submit, galaxy gave an error information like that: *Server Error:* An error occurred. See the error logs for more information. (Turn debug on to display exception reports here) I don't know where to find the error logs and how to turn debug on. Could you tell me how to fix the problem and upload the custom builds? Thanks a lot! Pengfei -- Pengfei Yu Center of Biophysics and Computational Biology University of Illinois Urbana-Champaign Illinois, 61820, US Cell phone: 217-898-6301 tel:217-898-6301 ___ 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://usegalaxy.org http://galaxyproject.org/wiki/Support ___ 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] Error on custom builds on public galaxy server
Hi Pengfei, The User - Custom Builds: form is used to set up genomes for use with Trackster. To use custom reference genomes with tools from the left tool panel, only the fasta dataset in the history is needed. It sounds like you already have loaded this, but if not, do this before following the steps below, using FTP to load if the genome is large: http://wiki.g2.bx.psu.edu/FTPUpload These are the steps to extract sequences with a custom genome: 1 - starting with the tool Fetch Sequences - Extract Genomic DNA 2 - set Fetch sequences for intervals in: to be the BED file of coordinates 3 - set Source for Genomic Data: to be History 4 - the form will refresh to reveal a new menu option, set Using reference file: to be the dataset from your history that is the fasta version of your reference genome 5 - set Output data type: to be FASTA It is important that the chromosome/scaffold names in the BED file are identical to the identifiers in the FASTA reference genome dataset. If there are mismatches, an error will result and corrections to the IDs should be made. Hopefully this helps, but if you continue to have problems, please send in a bug report (using the green bug icon) from the red failed Extract job and we can provide feedback. Best, Jen Galaxy team On 2/17/12 8:25 AM, Pengfei Yu wrote: Hi Jeremy, Thanks for the help! I can upload fasta data for custom builds (name:'NCI-H209-hg18'; key:'NCI-H209-V1') now. but when I try to use a bed file specific for this custom builds to fetch sequences from this custom genome, it gives error information that no sequences are available for 'NCI-H209-v1'. I might do something wrong, do you know what would be the reason? Thanks! Pengfei On Fri, Feb 17, 2012 at 7:40 AM, Jeremy Goecks jeremy.goe...@emory.edu mailto:jeremy.goe...@emory.edu wrote: Hi Pengfei and Noa, I suspect this was a temporary problem related to software issues that we encountered yesterday. Please try again and let us know if you're still having problems. Also, note that either a FASTA dataset or a Len file is needed, but not both. Using a FASTA dataset enables viewing genome data in Trackster. Best, J. On Feb 17, 2012, at 12:00 AM, Noa Sher wrote: I also had exactly the same problem yesterday - glad to see that maybe it wasn't just me. Happy for help! Noa On 16/02/2012 22:24, Pengfei Yu wrote: Dear sir or madam, I am using galaxy public server and I am trying to build a custom genome using the function custom Builds in user section. I specified the Name, Key and uploaded the genome fasta file also the chromosome length file. But when I click submit, galaxy gave an error information like that: *Server Error:* An error occurred. See the error logs for more information. (Turn debug on to display exception reports here) I don't know where to find the error logs and how to turn debug on. Could you tell me how to fix the problem and upload the custom builds? Thanks a lot! Pengfei -- Pengfei Yu Center of Biophysics and Computational Biology University of Illinois Urbana-Champaign Illinois, 61820, US Cell phone:217-898-6301 tel:217-898-6301 ___ The Galaxy User list should be used for the discussion of Galaxy analysis and other features on the public server atusegalaxy.org http://usegalaxy.org/. Please keep all replies on the list by usingreply 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 atusegalaxy.org http://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/ -- Pengfei Yu Center of Biophysics and Computational Biology University of Illinois Urbana-Champaign Illinois, 61820, US Cell phone: 217-898-6301 ___ 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
