- galaxy-user - lists.galaxyproject.org

Removal of duplicates and Cufflinks usage
by Lizex Husselmann 04 Nov '11

04 Nov '11

Hi All I've started analyzing my RNA-Seq data for two time points: Day0 and Day4 for control and treated. I've done aligning the data to the reference genome using Tophat. I've removed duplicates from the data sets. Could somebody please tell me, how important is it to remove duplicates and how will it influence my results if I don't remove? I want to start with Cufflinks all the way through to Cuffdiff. Where do I start since there are just so many options (in the manual) to choose from? What do I look for? Kind regards Lizex Disclaimer This message is confidential and may be covered by legal professional privilege. It must not be read, copied, disclosed or used in any other manner by any person other than the addressee(s). Unauthorised use, disclosure or copying is strictly prohibited and may be unlawful. The views expressed in this email are those of the sender, unless otherwise stated. If you have received this email in error, please contact ARC Service Desk immediately. (mailto:Servicedesk@arc.agric.za) To report incidents of fraud and / or corruption in the ARC use our Ethics Hotline by: Phone number : 0800 21 20 56 Fax number : 0800 200 796 Email address : fraud(a)kpmg.co.za For more information on the ARC Ethics Hotline, please visit our website at www.arc.agric.za.

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Re: [galaxy-user] Help with analysis
by Alex Daffornel 04 Nov '11

04 Nov '11

> > Brand new galaxy user here. > > I ran an RNA-seq Illumina experiment in which I compare cells from wild > type animals and cells from animals that have a deletion in a splicing > factor. Now I have my data in fastq format and need to do analysis to > figure out which transcripts are changed and how (see below). Problem is, I > have no idea whatsoever what to do. > > Can someone be so kind and write down a basic outline of analysis to > follow? > > My understanding from what I've been reading online is that once you have > fastq files you > > 1. Use FASTQ Groomer to convert to Sanger format > > 2. Evaluate the quality with FASTQ Summary Stats (and get boxplot of data) > > 3. Trim reads if their quality doesn't look good > What is considered "ok" quality? A score above 20? Is that the > mean score or the absolute score? How do I trim based on score only those > reads that have a low q score? (can I?) > > 4. Map the reads > What mapping software do you reccomend? BWA or Bowtie? Or Tophat? > What next? > Let's say that anything after the trimming is very fuzzy. > > > The questions I am interested in are > - What transcripts are upregulated / downregulated in mutant vs control ? > (I have 3 replicates of each) > - Are there introns that are retained in mutant (but not or less in > control)? > - Are there exons that are excluded in mutant (basically, I want to at > patterns of alternative splicing..) > > > Sorry for the very long message, but I have no idea who else to ask. > > Thanks!! > > > > > > >

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cufflinks error
by jh yu 04 Nov '11

04 Nov '11

Dear all: Recently I am using cufflinks to analyze differential expression between different conditions, but when using cufflinks an error occurred: An error occurred running this job:cufflinks v1.0.3 cufflinks -q --no-update-check -I 1 -F 0.050000 -j 0.050000 -p 8 -g /galaxy/main_database/files/002/991/dataset_2991920.dat Error running cufflinks. [bam_header_read] EOF marker is absent. [bam_header_read] invalid BAM binary header (this However, when I used the same parameters to analyze another file, it worked well: 19,904 lines format: gtf, database: rhodRHA1 Info: cufflinks v1.0.3 cufflinks -q --no-update-check -I 1 -F 0.050000 -j 0.050000 -p 8 -g /galaxy/main_database/files/002/991/dataset_2991920.dat The only difference is the size of each file, the failed one input file is 23 G, while the succeeded one input file is 3.5 G, is the size causing failure? Thank you in advance. Best wishes! Sincerely, Jinhai YU Jinhai YU, Ph.D Candidate 010-64888521 Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing, 100101, China

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cufflinks set parameters
by dongdong zhaoweiming 04 Nov '11

04 Nov '11

Hi, It's the first time for me to use cufflinks in galaxy, two choices confused me as follows: Perform Bias Correction: No Yes Bias detection and correction can significantly improve accuracy of transcript abundance estimates. Set Parameters for Paired-end Reads? (not recommended): No Yes what is the Bias correction and how does it work? And for "set parameters for paired-end reads? (not recommended)" ,what would be important difference between recommended and not recommended? Thanks a lot! dongdong

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data format
by Klaudyna Borewicz 04 Nov '11

04 Nov '11

Hi, I would like to use Galaxy to run LEfSe, but I don't know how to get the data into tabular format that is required (http://huttenhower.org/galaxy/tool_runner?tool_id=LEfSe_for) My data is 454 fasta files that I was analyzing with RDP to get the classification. It works fine, I get .txt file that i can load to Galaxy, it looks like this: norank Root 37646 unclassified_Root 9 domain Bacteria 37637 unclassified_Bacteria 5998 phylum OD1 0 unclassified_OD1 0 genus OD1_genera_incertae_sedis 0 phylum BRC1 0 unclassified_BRC1 0 genus BRC1_genera_incertae_sedis 0 phylum Deferribacteres 0 unclassified_"Deferribacteres" 0 class Deferribacteres 0 unclassified_Deferribacteres 0 order Deferribacterales 0 unclassified_Deferribacterales 0 family Deferribacterales_incertae_sedis 0 unclassified_Deferribacterales_incertae_sedis 0 genus Caldithrix 0 family Deferribacteraceae 0 unclassified_Deferribacteraceae 0 genus Calditerrivibrio 0 genus Mucispirillum 0 ..... but i need to have the labels in a hierarchical organization and I cannot find the way to get it to work. Please let me know if you have any suggestions, or maybe RDP is just not the way to go. Thank you and hope to hear from you soon, Klaudyna -- ____________________________________________________ Klaudyna Borewicz M.Sc, B.Sc Department of Veterinary and Biomedical Sciences University of Minnesota 1971 Commonwealth Ave. St. Paul, Minnesota 55108 Phone: (612)624-6226 FAX: (612)625-5203

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fastx reverse compliment failed - gzip: stdout: Broken pipe
by Lucinda Lawson 03 Nov '11

03 Nov '11

Hi all, We are running Galaxy on an Ubuntu 11.10 computer (5 TB, stripped, etc.). We are assembling a small genome (110 Gb). Our dataset isn't directly uploaded, but is accessed from a directory (if that matters). Everything went fine through the FASTQ Groomer, but when we ran Reverse-Compliment, we got the following error: fastx_reverse_complement: writing quality scores failed: File too large gzip: stdout: Broken pipe Any help that you might have would be greatly appreciated! Thanks! -Lucinda

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Downloading files
by KNIGHT M.R. 02 Nov '11

02 Nov '11

Is there any way of downloading processed files by e.g. FTP, rather than "Save as" in the web browser? I have a large (9.6Gb) BAM file to download, and downloading it via the browser seems unstable for some reason. Thanks, Marc.

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Re: [galaxy-user] Dataset not appearing as available input to SAM analyses
by Jennifer Jackson 02 Nov '11

02 Nov '11

Hi Clare, Galaxy is fairly transparent, so this occurs sometimes and can mean that either the server is very busy or there is another problem, perhaps with the dataset. Please note that mpileup is not supported yet, just pileup, although this is on our short list of to-do upgrades. If you used mpileup, this could very likely be the root cause of the tool error ("server error"). You can track the upgrade to mpileup through this ticket: https://bitbucket.org/galaxy/galaxy-central/issue/524 To troubleshoot a pileup file: 1 - double check the format of your data again the specification as outlined on the tool form for "NGS: SAM Tools -> Generate pileup from BAM dataset". 2 - try running the file type assignment again the job again to double check against a transient error. If pileup was used and the cause of the error is still unclear, we take a look we can provide feedback. Please share a link to your history using "Options -> Share or Publish" from the top of the history panel, generate a share link, then email that back to me directly. Please note which dataset presents the problem and confirm that "samtools pileup" was used. Thanks! Jen Galaxy team On 11/2/11 8:12 AM, James Nelson wrote: > Thanks Jen, but the very first time I tried changing the type to "pileup", Galaxy came back with > > Server Error > An error occurred. See the error logs for more information. (Turn debug on to display exception reports here) > > I somehow don't think the user's supposed to see the "error logs" or exception reports, so what next? > > Clare > > ----- Original Message ----- > From: "Jennifer Jackson"<jen(a)bx.psu.edu> > To: "James Nelson"<jcn(a)k-state.edu> > Cc: galaxy-user(a)bx.psu.edu > Sent: Wednesday, November 2, 2011 8:11:09 AM > Subject: Re: [galaxy-user] Dataset not appearing as available input to SAM analyses > > Hello Clare, > > Perhaps the loaded pileup datasets are not assigned to the "pileup" > datatype? Click on the pencil icon in the right upper corner of the > loaded pileup dataset, then use "Edit Attributes -> Change data type". > > Hopefully this helps, > > Best, > > Jen > Galaxy team > > > On 10/29/11 4:10 PM, James Nelson wrote: >> Hi, I'm trying to use the SAM/Filter pileup analysis on some pileup output files I've uploaded. But when I click the analysis link, these output files don't appear in the Select dataset dropdown menu; instead only one file, the output from FASTQ Summary Statistics which is obviously not pileup output, is shown. >> >> Thanks >> Clare Nelson >> ___________________________________________________________ >> 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

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Galaxy tracker browser freezes when zoomed in
by Steve 02 Nov '11

02 Nov '11

Hi, I'm going through the RNA-seq exercise at http://main.g2.bx.psu.edu/u/jeremy/p/galaxy-rna-seq-analysis-exercise. I'm at step 2 of mapping the reads where I've run tophat and have loaded the output files and the regGene tracks onto a new track browser. When I zoom in (to ~45kbp window), the accepted hits and one of the splice junction tracks from tophat do not show data and instead show a crossed pattern and the browser no longer allows clicking and dragging to move the window. Please advise. Cheers, Steve

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Removing low quality reads
by Getiria Onsongo 02 Nov '11

02 Nov '11

Galaxy Users, I would like to filter a .bam file to remove reads with low mapping quality, especially ambiguously mapped reads (MAPQ = 0). I can easily do this using the command line version of samtools as shown below. samtools view -bq 20 hba1.bam > hba1_MAPQ20.bam None of the options available under "NGS:SAM Tools" (e.g., Generate pileup and Filter SAM) provide an option for removing reads with low mapping quality. The history shown in http://main.g2.bx.psu.edu/u/onsongo/h/obtaininghighqualityreads shows the results I would like to obtain. Data 2 shows the results of Picard tools SAM/BAM Alignment Summary Metrics<http://main.g2.bx.psu.edu/tool_runner?tool_id=PicardASMetrics> on hba1.bam which contains reads with MAPQ values less than 20. As shown in this summary html, PF_READS_ALIGNED = 775 and PF_HQ_ALIGNED_READS = 241. Data 4 shows the results of Picard tools SAM/BAM Alignment Summary Metrics<http://main.g2.bx.psu.edu/tool_runner?tool_id=PicardASMetrics> on hba1_MAPQ20.bam which contains only reads with MAPQ greater than or equal to 20. As shown in this summary html, PF_READS_ALIGNED = 241 and PF_HQ_ALIGNED_READS = 241. Is there a way in Galaxy to filter a bam file to remove low quality mapped reads similar to using the samtools command line alternative shown above? Thanks, Getiria -- Getiria Onsongo, Ph.D. Bioinformatics Research Scientist Masonic Cancer Center, University of Minnesota Minneapolis, MN 55455 Phone: 612-625-0101

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