July 2013 - galaxy-user - lists.galaxyproject.org

HCC Mutations Workflow on Local Galaxy Instance
by Moritz Juchler 30 Jul '13

30 Jul '13

Hello, I am Moritz Juchler from University Heidelberg. For my Bachelor thesis I have to setup Galaxy to find SNP's in genomes from hcc patients. I have a 64-bit openSuse 11.3 server on which I installed Galaxy locally, since we have a) very large files (>30GB per patient) and b) the data is protection sensitive. I have to run this pipeline: http://www.nature.com/ng/journal/v44/n6/extref/ng.2256-S1.pdf (page 2) from this paper: http://www.nature.com/ng/journal/v44/n6/pdf/ng.2256.pdf I have some paired end data from patients, and I want to reproduce the pipeline as similar as possible. I have this so far: https://main.g2.bx.psu.edu/u/mj--/w/ngs I would be glad to even do 2-3 steps, I wont need much more for my thesis. But I find it so hard to find any information about what to do in Galaxy in practice. The first step in the workflow of the paper I included are the statistics on page 1 of the supplements, but those aren't necessary (?). So the first step I have to do after the alignment and the sam to bam conversion and the dedupe is the first step on page 2 of the supplements: "Variant calling Tumor" Which tool in Galaxy do I have to use in order to do this and the following steps? Any hints, links to papers or answers are welcome :) I asked the Galaxy Users Mailing list 1 week ago but unfortunately I didnt get any answers. Best Moritz

1 0

Problem with SAM-to-BAM conversion
by Fabrice BESNARD 29 Jul '13

29 Jul '13

Hello, I am trying to map Illumina paired-ends reads from the nematode Caenorhabditis briggsae. Here are the steps of my history: 1)I uploaded and groomed two .fq files corresponding to paired-end reads. In the attributes, I choose the cb4 database. 2)I mapped them using BWA (BWA Version: 0.5.9-r16 BWA run on paired-end data). However, I used the cb3 database here, since the cb4 was not available. 3)I filtered the output SAM file for mapped reads (galaxy tool version 1.0.0). cb3 is still said to be the reference database in the filtered SAM I got as output. 4) Then I tried to convert the SAM into BAM. Here it failed. When I chose the parameters of this tool, what I had for the 1st option "Choose the source for the reference list" is either: -from the history -locally cached I chose locally cached. Then, when running the tool, and I had this message error: "20: SAM-to-BAM on data 16: converted BAM error An error occurred with this dataset: Samtools Version: 0.1.18 (r982:295) No sequences are available for build (cb3), request them by reporting this error." As recommended, I report you now this error ! I found a similar problem in the Galaxy_user mailing list... But I did not get how the problem was solved ! http://user.list.galaxyproject.org/SAM-to-BAM-conversion-problem-td4135498.… I tried to change the attributes of the SAM and filtered-SAM files to cb4, but it did not work. Note also that in the error report, the version is said to be 0.1.18. But when I click on the SAM-to-BAM tool, the header of the page says: SAM-to-BAM (version 1.1.2). I don't know if this matters or not. Could you please help me fixing this issue ? Many thanks ! -- Fabrice Besnard Institute of Biology of the Ecole Normale Supérieure (IBENS) 46 rue d'Ulm, 75230 Paris cedex 05, France 8th floor. Office: Room 802. Lab: Room 817. mail: fbesnard(a)biologie.ens.fr Tel: +33-1-44-32-39-44

2 3

Illumina Methylation tool in toolshed
by Sachit Adhikari 29 Jul '13

29 Jul '13

Hi, This is my third post regarding this matter but however I didn't get any reply. I am trying to find a tool for r Illumina Methylation. Is there any available on the tool shed or test tool shed? Thanks, Sachit

2 3

Launching IGB from Galaxy
by Seth Stern 25 Jul '13

25 Jul '13

Hi, Ive been trying to launch the IGB viewer by clicking the Local button on the SAM-to-BAM converter (in History). (I have a local version of the viewer running on my computer.) But instead of transferring data to the viewer (what I expected to happen), it launches the web browser with this address: http://localhost:7085/UnibrowControl?version=?&feature_url_0=http://main.g2. bx.psu.edu/display_application/bbd44e69cb8906b5ec213d3ada68caac/igb_bam/Loca l/1ce2985e52430eb3/data/SAM_to_BAM_on_data_10_and_data_20__converted_BAM_bbd 44e69cb8906b5ec213d3ada68caac.bam&sym_name_0=SAM_to_BAM_on_data_10_and_data_ 20__converted_BAM&sym_method_0=http://main.g2.bx.psu.edu/display_application /bbd44e69cb8906b5ec213d3ada68caac/igb_bam/Local/1ce2985e52430eb3/data/SAM_to _BAM_on_data_10_and_data_20__converted_BAM_bbd44e69cb8906b5ec213d3ada68caac. bam&query_url=http://main.g2.bx.psu.edu/display_application/bbd44e69cb8906b5 ec213d3ada68caac/igb_bam/Local/1ce2985e52430eb3/data/SAM_to_BAM_on_data_10_a nd_data_20__converted_BAM_bbd44e69cb8906b5ec213d3ada68caac.bam&server_url=ga laxy Can anyone help me understand what is going on ? Thanks, Seth Seth Stern, Ph.D. sethster(a)comcast.net 650 996 8726 (cell)

2 1

Galaxy error
by Toni Delorey 25 Jul '13

25 Jul '13

Hello, I'm trying to convert a .bed file to a FASTA file. I get the following error when I do. "An error occurred with this dataset: *No sequences are available for 'sacCer2', request them by reporting this error.*" I'm not sure what the issue is? Thanks, Toni -- Toni Marie Delorey Regev Lab, 6175-OO The Broad Institute of MIT and Harvard 7 Cambridge Center Cambridge, MA 02141 Email: delorey(a)broadinstitute.org Phone: 617-714-8225

3 4

analysing pooled data with FreeBayes
by Nicola Smith 24 Jul '13

24 Jul '13

Hi, I am new to this and I hope someone can help. I have pooled sequencing data that I am trying to analyse using Galaxy. I've done quite a bit of online searching and it seems that FreeBayes should be able to do this, if I select "set population", click the "Assume that samples result from pooled sequencing" option and change the ploidy to nx2 (number of alleles, where n is the number of subjects and the organism is diploid). However, whenever I do this I get an error: usually just "Killed" I was originally setting my polidy rather high (190 as I have 95 subjects pooled), so I wondered if this was the problem, however, it fails if I do a ploidy of only 4 too. I've tried various things to try to see where I am going wrong: All with the same BAM file: Set population model options: Do not set --> works Set population model options: set, Assume that samples result from pooled sequencing: not ticked, Default ploidy for the analysis: 2 --> works Set population model options: set, Assume that samples result from pooled sequencing: ticked, Default ploidy for the analysis: 2 --> works Set population model options: set, Assume that samples result from pooled sequencing: ticked, Default ploidy for the analysis: 4 --> fails (killed) Set population model options: set, Assume that samples result from pooled sequencing: ticked, Default ploidy for the analysis: 10 --> fails (killed) Set population model options: set, Assume that samples result from pooled sequencing: ticked, Default ploidy for the analysis: 10 --> fails (killed) It seems that it is the ploidy part that I am doing wrong, as it works with pooled data but ploidy of 2. I'm sure I have to change the ploidy though, or else how does the program know how many subjects are in the pool? Also, everywhere that I've ready says you have to change the ploidy. I apologise if my question is naive. As I said, I am new to Galaxy and this is the first thing I am trying to do! Any help / suggestions would be appreciated, Thanks, Nic

3 2

Rename tool output file in Galaxy
by Sachit Adhikari 24 Jul '13

24 Jul '13

Hi group, The galaxy stores the output of the job on files/043/dataset_*ID*.dat. I have two questions here.: 1) Can I find the ID of my output from the Galaxy history? I tried Edit Attributes, Annotations, tags but couldn't find it. 2) Can I rename my output while initiating the task? If I can how can I do this and what are the consequences. Thank you. Regards, Sachit

3 7

Cuffdiff2
by Anto Praveen Rajkumar Rajamani 23 Jul '13

23 Jul '13

Hello, I am new to Galaxy. I plan to conduct RNA-seq data analysis using Galaxy. I find that the public Galaxy server has a very old version of Cuffdiff 0.0.5. Does any Galaxy server have updated Cuffdiff2? If I buy aws cloud space, will I get access to the updated software? Thank you. Best wishes, Anto

2 1

"Significant" in Cuffdiff's ouput
by Hoang, Thanh 22 Jul '13

22 Jul '13

Hi all, I have some questions about how Cufffdiff does the statistical analysis. I am looking for DE genes in two sample groups ( 3 replicate per group). In the Cuffdiff;s gene_exp.diff, I found many genes that have very large RPKM fold-change between two groups (with p value < or > 0.05) but still NO significant. Something like this: 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 ENSMUSG00000047139 ENSMUSG00000047139 Cd24a 10:43579168-43584262 q1 q2 OK 96.2585 2700.55 4.8102 1.6486 0.03995 0.078237 no ENSMUSG00000066975 ENSMUSG00000066975 Cryba4 5:112246492-112252518 q1 q2 OK 424.582 46190.2 6.7654 0.598327 0.3408 0.442128 no Then I checked the READ_GROUP_TRACKING file for those genes to check the RPKM value for each replicate: tracking_id condition replicate raw_frags internal_scaled_frags external_scaled_frags FPKM effective_length status ENSMUSG00000047139 q1 1 11256 5876.82 5876.82 125.915 - OK ENSMUSG00000047139 q1 0 3783 4343.44 4343.44 42.0316 - OK ENSMUSG00000047139 q1 2 10051 5639.48 5639.48 120.829 - OK ENSMUSG00000047139 q2 1 76771 156059 156059 3343.66 - OK ENSMUSG00000047139 q2 0 82394 162172 162172 1420.33 - OK ENSMUSG00000066975 q1 1 12825 6696 6696 407.899 - OK ENSMUSG00000066975 q1 0 3694 4241.26 4241.26 375.211 - OK ENSMUSG00000066975 q1 2 14397 8077.95 8077.95 490.636 - OK ENSMUSG00000066975 q2 1 348103 707619 707619 42455.1 - OK ENSMUSG00000066975 q2 0 420896 828430 828430 48920.6 - OK ENSMUSG00000066975 q2 2 331098 767405 767405 47195 - OK Would not I expect these DE genes are significant? Could anyone explain why Cufflinks show this result? Best regards Thanh

2 1

Trackster Error
by Seth Stern 22 Jul '13

22 Jul '13

Hi, I'm trying to visualize a BWA mapping using Trackster, but I'm getting this error: Trackster Error needLargeMem: trying to allocate 0 bytes (limit: 100000000000) Can anyone explain to me what is going on and possibly how to fix it ? Thanks, Seth Seth Stern, Ph.D. sethster(a)comcast.net 650 996 8726 (cell)

2 1