- galaxy-user - lists.galaxyproject.org

Tophat mapping and Cufflinks output issues
by Jim Cohen 16 Jan '13

16 Jan '13

Hello Galaxy Users- I've been using the Main Galaxy server to work on an RNA-Seq project for a non-model plant, and I've noticed that my output from Tophat and Cufflinks might not be as good as I'd like. I have a reference transcriptome assembled in Trinity, and it is based on the same Illumina-generated 100 bp reads I'm trying to map to it. When I use Tophat to map the reads to the reference transcriptome (I have trimmed the reads and filtered the lower quality ones), only about 10% of the reads actually map, so I go from 30,000,000 reads before mapping to 3,000,000 that are actually mapped. Therefore, I feel like I'm losing a lot of data. When I've changed the parameters to allow for more mismatches, not many more reads seem to map, and in many cases, the Tophat run fails and I receive the error message: "*Settings: Output files: "/tmp/ 3030460.cyberstar.psu.edu/tmpWbxTnm/dataset_5530451.*.ebwt" Line rate: 6 (line is 64 bytes) Lines per side: 1 (side is 64 bytes) Offset rate: 5 (one in 32) FTable chars: 10 Strings: unpacked Max bucket size: def"*. I've had similar numbers of reads map with Bowtie by itself and BWA as well. I've also tried mapping the reads to the assembled isoforms (contigs) of the transcriptome, and this results in many more reads (close to 90%) being mapped. Therefore, I figure the reads should map to the reference transcriptome, and I'm not sure why this isn't happening. The other issue I've run into is that in Cuffdiff only about 4,800 genes appear in the output files as being tested for differential expression. There are approximately 100,000 genes in the reference transcriptome, so I was thinking that there should be more than ca. 4,800 that are tested for differential expression. Should each gene be tested? Does Cuffdiff just not report some of the genes that are not differentially expressed, or is the program not testing all of the genes? If anyone can provide some help, guidance, or a suggestion, I'd greatly appreciate it. Thanks, and take care. Jim

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identify different number of differential expressed genes using ensemble or reseq GTF
by Wei Liao 15 Jan '13

15 Jan '13

Hi all, I am analyzing significant differential expressed genes for a pair of normal V.S tumor, using Cuffdiff 2.0.2. I noticed that by using ensemble GTF and refseq GTF, the results showed a big difference on the number of genes being significant expressed. For ensemble GTF, there are only 250 genes differential expressed. But for refseq GTF, there are about 1000 genes. I am running these data on Galaxy server and with the same workflow. Can anyone explain what is going on here? so which result should I trust? Thanks. -- Wei Liao Research Scientist, Brentwood Biomedical Research Institute 16111 Plummer St. Bldg 7, Rm D-122 North Hills, CA 91343 818-891-7711 ext 7645

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Re: [galaxy-user] converting solid files
by Enis Afgan 14 Jan '13

14 Jan '13

Hi Nuphar, I believe yu should be using a tool that is color-space aware and not converting the data - Galaxy Main has 'Bowtie for SOLID' available, which should understand your data as is. BTW, for these types of questions, you should send the email to galaxy-user mailing list (http://lists.bx.psu.edu/listinfo/galaxy-user) which I have CC'd now, because that will get your question much more exposure and thus higher likelihood of a useful answer. Cheers, Enis On Sun, Jan 13, 2013 at 9:58 PM, Nuphar Salts <nupharsalts(a)gmail.com> wrote: > Hello, > > We're trying to convert csfasta and qual files to fatsq, using galaxy. > The problem is that it seems that A is always used as the first base, for > each number representing a color. > We want to use bowtie for analyzing RNA-seq results. > Is it possible to convert our files with galaxy or should we use another > tool? > > Thanks, > Nuphar. >

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Uploading files to history via API
by Neil.Burdett＠csiro.au 14 Jan '13

14 Jan '13

Hi, It seems the API documentation is a bit scarce. I'd like to upload a file from the command line using the API rather than the GUI, but would like to see the file in the history. Does anyone have any information how to do this/ any tools that are available. Thanks Neil

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Galaxy January 11, 2013 Distribution & News Brief
by Jennifer Jackson 11 Jan '13

11 Jan '13

*Galaxy January 11, 2013 Distribution & News Brief* <http://wiki.galaxyproject.org/News/2013_01_11_DistributionNewsBrief> * Complete News Brief <http://wiki.galaxyproject.org/DevNewsBriefs/2013_01_11>* * Highlights:* * /*GCC2013 Training Day Topic Nomination closes TODAY!*/ Read more... <http://wiki.galaxyproject.org/News/GCC2013TrainingDayNominations> or see http://wiki.galaxyproject.org/Events/GCC2013/TrainingDay * XML defined Tool shed repository dependencies <http://wiki.galaxyproject.org/DefiningRepositoryDependencies> introduced * Various enhancements <http://wiki.galaxyproject.org/DevNewsBriefs/2013_01_11#Other_Tool_Shed> to Tool Shed <http://wiki.galaxyproject.org/Tool%20Shed> containers, display of README files and SVG images, and the tool shed's functional test framework <http://wiki.galaxyproject.org/HostingALocalToolShed#Functional_test_framewo…>. * New genomes <http://wiki.galaxyproject.org/DevNewsBriefs/2013_01_11#Data> added to *Main <http://wiki.galaxyproject.org/Main>* Galaxy instance and rsync download. * Plus bug fixes and related enhancements for visualizations, histories, workflows, and tools. * http://getgalaxy.org* *http://bitbucket.org/galaxy/galaxy-dist* *http://galaxy-dist.readthedocs.org* new: $ hg clone http://www.bx.psu.edu/hg/galaxy galaxy-dist upgrade: $ hg pull -u -r a4113cc1cb5e / *Thanks for using Galaxy,* / The Galaxy Team <http://wiki.galaxyproject.org/Galaxy%20Team> -- Jennifer Hillman-Jackson Galaxy Support and Training http://galaxyproject.org

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macs error for chipseq data
by Sun, Wenping [USA] 11 Jan '13

11 Jan '13

Dear galaxy users, I've encountered the error while running macs on galaxy for chipseq data. I used fastq groomed files as input and had the following error An error occurred running this job: /bin/sh: macs: not found Anybody can kindly provide some hints? Thanks, Kathryn

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where to submit galaxy error messgae
by Jennifer Jackson 11 Jan '13

11 Jan '13

Hi Kathryn, If you encounter an error while using the public Main Galaxy server at http://main.g2.bx.psu.edu (usegalaxy.org) the first thing to do is review the tool usage help (on tool form) and the input dataset formats. Often the error message or 'i' info page's standard out/error will have some details about the problem, or the info revealed when clicking on the 'bug' icon (this doesn't automatically send in a bug report, you can also just review it). Many problems can be solved with minor adjustments, some tips are in our wiki here: http://wiki.galaxyproject.org/Support#Help.21_Common_Solutions But if you are still stuck, then please submit the error as a bug report, being sure to leave the inputs and error itself undeleted in your history (this is important), following this method: http://wiki.galaxyproject.org/Support#Reporting_tool_errors When running your own instance, or a cloud instance, other factors can contribute to problems, including the instance administration options and set-up. It is best to send these types of questions, with clear descriptions of the issues and some description of the troubleshooting you have done (this tends to generate the most feedback) to the dedicated development mailing list at galaxy-dev(a)bx.psu.edu. This will give it the best exposure to the dev community. http://wiki.galaxyproject.org/Support#Mailing_Lists I see your other question now posted about MACS. At first glance this looks like a local install tool configuration problem, which would be a good fit for the galaxy-dev list, but we will respond to that question or move it over there and then respond. We do our best not to cross/double post to both lists. Hopefully this helps explain things a bit more! All list emails come to the same folks here on the core team - this org just helps us to track. But, the real benefit is for our community, so they can participate in their area of interest and more effectively search prior Q & A. Take care, Jen Galaxy team On Jan 10, 2013, at 2:31 PM, "Sun, Wenping [USA]" <Sun_Wenping(a)bah.com> wrote: > Hi Jennifer, > > I have few messages with the error that I had while running galaxy modules. Is galaxy-user is good email for sending and getting the responses? > > Thank you very much, > Kathryn Jennifer Hillman-Jackson Galaxy Support and Training http://galaxyproject.org

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BWA and FASTQ Joiner issues
by Hilde Stawski 10 Jan '13

10 Jan '13

Hello, I hope someone might be able to help me with these issues, as I'm relatively new at Bioinformatics. When analyzing data on the main website of Galaxy (all samples from the same Illumina MiSeq run) some sets fail in the BWA alignment. I have tried rerunning my workflow again, reuploading the FastQ files (in case they were corrupted) but BWA fails every single time. I've pasted the error log below. Q1: Why are most of my datasets aligned by BWA without a problem, but some consistently fail? So, some people at SEQanswers suggested I install a local Galaxy client on my computer, and I'm now trying to rework my workflow so I can use BFAST as an aligner. BWA was giving us some trouble due to indels in our mtDNA sequence, so we are trying to find another aligning tool that is more capable of working with these indels, anyway. However, now I'm running into a second problem. I have PE data, and after grooming both FASTQ files, the FASTQ Joiner generates an empty file. I did try the workaround mentioned here ( http://lists.bx.psu.edu/pipermail/galaxy-user/2012-April/004519.html) but I'm not sure I understand the following instructions "NGS: QC and manipulation -> Tabular to FASTQ" run twice Recreate both FASTQ files from the same tabular file." I ran FASTQ to Tabular with two columns, and joined the files on the first column. Why/how should I recreate both FASTQ files if I wanted to create just one single file to use as input for alignment? FASTQ Groomer doesn't seem to be able to groom the data either "Based upon quality and sequence, the input data is valid for: None Input ASCII range: '0'(48) - 'N'(78) Input decimal range: 15 - 45" Q2: How do I create a single file as input for the aligner? Thanks in advance for any help, Hilde --- The alignment failed. Error generating alignments. [bwa_sai2sam_pe_core] convert to sequence coordinate... [infer_isize] (25, 50, 75) percentile: (2402, 5234, 8910) [infer_isize] low and high boundaries: 151 and 21926 for estimating avg and std [infer_isize] inferred external isize from 251269 pairs: 5940.068 +/- 4124.607 [infer_isize] skewness: 0.511; kurtosis: -0.744; ap_prior: 1.00e-05 [infer_isize] inferred maximum insert size: 23222 (4.19 sigma) [bwa_sai2sam_pe_core] time elapses: 1.37 sec [bwa_sai2sam_pe_core] changing coordinates of 0 alignments. [bwa_sai2sam_pe_core] align unmapped mate... [bwa_paired_sw] 3297 out of 10352 Q17 singletons are mated. [bwa_paired_sw] 0 out of 188377 Q17 discordant pairs are fixed. [bwa_sai2sam_pe_core] time elapses: 2241.09 sec [bwa_sai2sam_pe_core] refine gapped alignments... 1.37 sec [bwa_sai2sam_pe_core] print alignments... 1.76 sec [bwa_sai2sam_pe_core] 262144 sequences have been processed. [bwa_sai2sam_pe_core] convert to sequence coordinate... [infer_isize] (25, 50, 75) percentile: (2595, 7186, 11297) [infer_isize] low and high boundaries: 151 and 28701 for estimating avg and std [infer_isize] inferred external isize from 84557 pairs: 7236.733 +/- 4785.357 [infer_isize] skewness: 0.066; kurtosis: -1.262; ap_prior: 1.00e-05 [infer_isize] inferred maximum insert size: 27144 (4.16 sigma) [bwa_sai2sam_pe_core] time elapses: 0.38 sec [bwa_sai2sam_pe_core] changing coordinates of 0 alignments. [bwa_sai2sam_pe_core] align unmapped mate... [bwa_paired_sw] 482 out of 3212 Q17 singletons are mated. [bwa_paired_sw] 0 out of 40389 Q17 discordant pairs are fixed. [bwa_sai2sam_pe_core] time elapses: 532.50 sec [bwa_sai2sam_pe_core] refine gapped alignments... /bin/sh: line 1: 28068 Segmentation fault bwa sampe /tmp/ 3030216.cyberstar.psu.edu/tmprNNXwj/tmpXybcjA /tmp/ 3030216.cyberstar.psu.edu/tmpq3YCcl/tmpKvAb8e /tmp/ 3030216.cyberstar.psu.edu/tmpq3YCcl/tmpvUAE3E/galaxy/main_pool/pool3/files/… /galaxy/main_pool/pool3/files/005/540/dataset_5540837.dat >> /galaxy/main_pool/pool2/tmp/job_working_directory/004/860/4860532/galaxy_dataset_5540842.dat

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Re: [galaxy-user] Download data using FTP-access?
by Jennifer Hillman-Jackson 09 Jan '13

09 Jan '13

Hi Karen, I don't run windows OS, but found this link that seems to describe the same situation with a solution. Maybe give it a try and see if it works? http://stackoverflow.com/questions/3562095/using-curl-to-get-a-https-webpag… This link also looked helpful, just from a set-up perspective and since your curl install is new, but I don't know if you are using a 64-bit machine or not: https://support.gradesfirst.com/forums/20230492-how-to-setup-curl-on-window… I found these with a google using "curl windows 7 https" - there are other hits, but these two looked the most promising as a first go for troubleshooting. Good luck! Jen Galaxy team On 1/9/13 1:46 AM, Karen Margrethe Jessen wrote: > Hi Jennifer, > > Thanks for your answer. I have downloaded the Curl program and I am trying to use it from commando promt in Windows 7. Hovewer, when typing the commando: > > ’C:\ curl https://xxxxxxxxxxxxxxxxxxx’ > > in commando promt, I get the following message: > > ’Protocol https not supported or disabled in libcurl’. > > How do I make Curl download from https? > > Karen > > > ________________________________________ > Fra: Jennifer Hillman-Jackson [jen(a)bx.psu.edu] > Sendt: 8. januar 2013 16:33 > Til: Karen Margrethe Jessen > Cc: galaxy-user(a)lists.bx.psu.edu > Emne: Re: [galaxy-user] Download data using FTP-access? > > Hello Karen, > > From a shell/unix/terminal window on your side, use "curl" to download > datasets. > > The link can be obtained by right clicking the floppy disk icon inside a > history item and choosing "Copy Link Address" (for most datasets) or > "Download Dataset/Download bam_index" (for BAM datasets there are two > downloads). Once you have the link: > > $ curl -O '<link>' > > Hopefully this helps! > > Jen > Galaxy team > > > On 1/8/13 2:27 AM, Karen Margrethe Jessen wrote: >> Is there any way of downloading processed files at galaxyproject.org by >> e.g. FTP, instead of “Save as” in the web browser? I have some large >> (~50Gb) BAM files to download, and downloading it via the browser is not >> stable. >> >> Thanks, Karen. >> >> >> ___________________________________________________________ >> 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 Hillman-Jackson > Galaxy Support and Training > http://galaxyproject.org > -- Jennifer Hillman-Jackson Galaxy Support and Training http://galaxyproject.org

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Remove "unpaired" reads from quality-filtered pared-end fastq files.
by 柴田弘紀 09 Jan '13

09 Jan '13

Hi there, I obtained two fastq files from GA paired end run. I filtered each file by quality using fastq tool kit. Then some forward reads may be removed by low quality whereas the reverse counterparts are OK to be remained on the other file, or vice versa. I want to remove those "unpaired" reads from filtered fastq files so that the two new fastq files contain the identical sets of the reads. Is it possible to do it on galaxy? Thank you very much. Hiroki

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