[galaxy-user] Reference genome , fasta with or without gene feature
Dear All I am analysing bacterial RNA seq (illumina). as my reference genome is not in galaxy. so i need to downlaoded the reference genome from NCBI and upload to my history Should i use reference genome with gene features or single fasta file with out any annotation information. even after download should i need to modify or it should be used as it is. Thanks in advance Ateeq___ 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] Bacterial RNA seq analysis
Dear All I need some (lots) suggestions and help, first and most important is that i am working on bacterial RNA seq (illumina reads) my analysis steps are as following Step 1. FASTQ sequence data was groomed Step 2. I did mapping by Bowtie with default parameters. Reference genome fasta file i am using from my history, because the reference genome is not vaialble on galaxy. Step3. i sorted the bowtie output file using r workflow (Germy Goecks workflow) , link below https://main.g2.bx.psu.edu/u/jeremy/p/transcriptome-analysis-faq#faq2 Step4. this sorting provided me Concatenate files Step5. Concatenated files were used to run CUFFLINK, this provided me assembled trancript file Step6: Assembled transcript files from step 5 were used for CUFFMERGE Step 7: For CUFFDIFF Transcript GTF file generated from Step 6and concatenate files from step 4 were used Now my question is if this workflow is acceptable for bacterial transcriptome anaylsis, Should i filter SAM file, if yes then at which step Should i convert SAM file to the BAM file, then at which step it should be Is that Ok to use fasta of reference genome for mapping should it be converted to any other format, if yes then what should be the workflow If any one have experince of bowtie parametes to map bacterial RNA seqquence analsis are very much welcomed Thanking you all ___ 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] Cuffdiff
Dear All I have simple and question for cuffdiff should we run cuffdif on merge transcript file (produced by cuffmerge) and concatenate data sets or directly on cufflink produced files, in the later case, i have two transcript files resulting from cufflink on sample 1 and 2 respectively, result using sample 1 as transcripts are not the same when i am suing sample 2 as transcript i am bit confused what should be the correct way any help is very much welcomed Best ateeq Ateequr Rehman House No. 2 ground floor Blauenstr. 10 79115 Freiburg im Breisgau___ 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] Disk space quota Issue
Dear Administartor My disk space quote is full, is their any way to increase it,or only way is delete some files... Best Ateeq Ateequr Rehman House No. 2 ground floor Blauenstr. 10 79115 Freiburg im Breisgau ___ 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] Cuffdiff result P and q values
Dear galaxy user After running cuffdiff on my two samples (SAM files from bowtie) i got a list with p and q values, and löast colum is saying abou significance with P value, it seems like the comparison should be significant, but in Q value is 1, and last coumn is saying not significant any one have an idea, how to interpret it , should we take any comparsion with less than 0.05 p value as significant or not tables in excel looks like it Nay help is welcome best Ateeq test_id gene_id gene locus sample_1 sample_2 status value_1 value_2 log2(fold_change) test_stat p_value q_value significant CUFF.428.1 CUFF.428 - gi|190572091|ref|NC_010943.1|:1575813-1577629 q1 q2 NOTEST 171.773 605.136 -150.518 588.996 3,86E-05 1 no CUFF.462.1 CUFF.462 - gi|190572091|ref|NC_010943.1|:1680283-1681214 q1 q2 NOTEST 696.628 322.149 -111.266 538.062 7,42E-03 1 no CUFF.635.1 CUFF.635 - gi|190572091|ref|NC_010943.1|:2343969-2346219 q1 q2 NOTEST 396.469 223.951 -0.824027 476.902 1,85E-01 1 no CUFF.512.1 CUFF.512 - gi|190572091|ref|NC_010943.1|:1840464-1843486 q1 q2 NOTEST 136.314 70.604 -0.949109 422.322 2,41E-01 1 no CUFF.632.1 CUFF.632 - gi|190572091|ref|NC_010943.1|:2346561-2347408 q1 q2 NOTEST 351.508 167.567 -106.882 415.844 3,20E-01 1 no CUFF.941.1 CUFF.941 - gi|190572091|ref|NC_010943.1|:3664426-3665364 q1 q2 NOTEST 282.247 133.798 -10.769 412.254 3,75E-01 1 no CUFF.616.1 CUFF.616 - gi|190572091|ref|NC_010943.1|:2301552-2303180 q1 q2 NOTEST 169.682 744.885 -118.774 462.107 3,82E-01 1 no CUFF.617.1 CUFF.617 - gi|190572091|ref|NC_010943.1|:2295763-2297758 q1 q2 NOTEST 225.933 112.178 -101.011 454.517 5,49E-01 1 no CUFF.9.1 CUFF.9 - gi|190572091|ref|NC_010943.1|:41597-42402 q1 q2 OK 1729.08 2797.07 0.693913 -4.461 8,16E-01 0.000179474 yes CUFF.956.1 CUFF.956 - gi|190572091|ref|NC_010943.1|:3665445-3669232 q1 q2 NOTEST 518.525 323.653 -0.679966 444.565 8,76E-01 1 no CUFF.549.1 CUFF.549 - gi|190572091|ref|NC_010943.1|:2043111-2043664 q1 q2 OK 7148.23 11816.4 0.725138 -421.443 2,50E+00 0.000275446 yes CUFF.872.1 CUFF.872 - gi|190572091|ref|NC_010943.1|:3489557-3490326 q1 q2 NOTEST 220.274 840.662 -13.897 416.179 3,16E+00 1 no CUFF.636.1 CUFF.636 - gi|190572091|ref|NC_010943.1|:2348784-2352394 q1 q2 NOTEST 114.384 601.415 -0.927447 414.807 3,35E+00 1 no CUFF.605.1 CUFF.605 - gi|190572091|ref|NC_010943.1|:2271979-2275960 q1 q2 NOTEST 217.007 133.837 -0.697264 409.373 4,24E+00 1 no CUFF.568.1 CUFF.568 - gi|190572091|ref|NC_010943.1|:2160538-2164415 q1 q2 NOTEST 74.365 377.013 -0.980011 395.097 7,78E+00 1 no CUFF.597.1 CUFF.597 - gi|190572091|ref|NC_010943.1|:2250029-2250918 q1 q2 NOTEST 229.389 105.246 -112.403 386.937 0.000109116 1 no Ateequr Rehman House No. 2 ground floor Blauenstr. 10 79115 Freiburg im Breisgau___ 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] ILLumina 1.9 Hiseq
Dear Glaxy users and admin I ran my sequence data on FASTQC tool, output says it is EncodingSanger / Illumina 1.9 now i want to groom my file, but groomer does not have option for 1.9 in Input FASTQ quality scores type any idea which option i should select to grroom my file, later i want to run Bowtie or Tophat, Thanks___ 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] Cufflinks related problem
Dear Bomba I am bit confused , Are you running bowtie --- tophat Cufflink For bowtie run you are using reference genome annotation in gff3 format I am curremntly totally unable to figure it out correctly, how i should analyse my RNA seq data Best Ateeq From: Noa Sher noa.s...@gmail.com To: Bomba Dam bomba@visva-bharati.ac.in Cc: galaxy-user@lists.bx.psu.edu Sent: Wednesday, February 22, 2012 7:28 AM Subject: Re: [galaxy-user] Cufflinks related problem Hi Bomba I cant know for sure without seeing your files but I originally had the same problem and it ended up being because the way the genome was named in the fasta genome file was not the same as the way it was named in column 1 of my gtf file. I would check that first. Also- with bacteria you dont want to run cufflinks with default parameters- use the galaxy browser to check how your data looks after tophat and you will most likely see very strange gene-spanning introns. Change the max intron size to 1000-1500 and the min distnace to 101-5nt and you should get results that make much more sense. Good luck Noa On 22/02/2012 00:12, Bomba Dam wrote: Dear Noa, Can you please tell me the parameters that I should keep while mapping bacterial transcripts using cuffkins. I have kept the default parameters as in Cufflinks and used my custom genome annotation in gff3 format. The cufflinks seems to work Ok but all the FPKM values in these files are zero. As suggested by other users I have checked the correctness of my GFF3 files. The corresponding fasta file was used for mapping the transcripts using Bowtie. Are there any special trick for mapping bacterial transcriptome. regards, Bomba ___ 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] 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
[galaxy-user] genome for Stenotrophomans
Dear Galaxy AdministratorI am analysing transcriptome and genome of clinical isolatesStenotrophomonasmaltophilia, would be highly obliged if you could please upload Stenotrophomonas maltophilia K279 and Stenotrophomonas maltophilia R551-3 genome acccesion numbers at NCBI are as following Stenotrophomonas maltophilia K279 (Accession AM743169) Stenotrophomonas maltophilia R551-3, complete genome Accession: CP00 Thanking you all in advance Ateequr Rehman___ 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] data upload
Dear galaxy UsersI am very very new to galaxy, will be highly obliged if some one could help me to find the way to analyse bacterial tramnscriptome. in the first step itself , i am having trouble to upload files...Does any one knows how to generate URL to upload data, my fastq files are about 3 gb each, Is there any specific pipeline to analyse bacterial transcriptomeThanking all of youAteeqAteequr RehmanHouse No. 2 ground floorBlauenstr. 10 79115 Freiburg im Breisgau From: Dannon Baker dannonba...@me.com To: mailing list margeem...@gmail.com Cc: galaxy-user@lists.bx.psu.edu Sent: Wednesday, January 11, 2012 2:11 PM Subject: Re: [galaxy-user] CloudMan - Persist changes to file system is not a hyperlink / clickable For sharing a cluster (of any kind) just click the sharing icon ()next to the cluster name and the dialog that pops up will walk you through the next steps.-DannonOn Jan 11, 2012, at 7:27 AM, mailing list wrote:So how do I get a share string for /mnt/galaxyData?Thanks again,GregOn Tue, Jan 10, 2012 at 4:16 PM, Dannon Baker dannonba...@me.com wrote:Sorry, I should have specified before -- /mnt/galaxyData is automatically persisted, as it is assumed (and used behind the scenes in the case of a Galaxy Cluster) that this is where data will be kept. It's only /mnt/galaxyTools or /mnt/galaxyIndices that need individual flagging for persistence.So, /mnt/galaxyData *is* safe to use for your own customizations and will be persisted automatically with no additional steps required on your part.-DannonOn Jan 10, 2012, at 4:02 PM, mailing list wrote:I'm following the instructions here:http://wiki.g2.bx.psu.edu/Admin/Cloud/Customize%20Galaxy%20CloudI ssh'd in and just made this change:cd /mnt/galaxyDatasudo git clone git://github.com/JaneliaSciComp/msg.gitNow I want to persist it, but as my subject line says, I don't seem tohave the option in the admin.Thanks,Greg___The Galaxy User list should be used for the discussion ofGalaxy analysis and other features on the public serverat usegalaxy.org. Please keep all replies on the list byusing "reply all" in your mail client. For discussion oflocal Galaxy instances and the Galaxy source code, pleaseuse the Galaxy Development list:http://lists.bx.psu.edu/listinfo/galaxy-devTo 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 ofGalaxy analysis and other features on the public serverat usegalaxy.org. Please keep all replies on the list byusing "reply all" in your mail client. For discussion oflocal Galaxy instances and the Galaxy source code, pleaseuse the Galaxy Development list: http://lists.bx.psu.edu/listinfo/galaxy-devTo 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/
