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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

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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

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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

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Cuffdiff2
by Anto Praveen Rajkumar Rajamani 24 Jul '13

24 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

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"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

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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)

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Exceptionally high RPKM values of miRNA and other short genes in Cuffdiff's output
by Hoang, Thanh 19 Jul '13

19 Jul '13

Hi all, I have been analyzing my RNA-seq data on mouse tissues. My RNA-data is single-ended and 51 bp in length. I ran TopHat/Cufflink/Cuffdiff to test to differential gene expression In the Cuffdiff's output, I got very high RPKM value for some of miRNA and some other short genes ( less than 100bp). These genes are in the top genes with the highest RPKM. I think the RPKM values of these genes are probably too high to be true. *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* *ENSMUSG00000093077* *ENSMUSG00000093077* *Mir5105* *5:146231229-146302874* *Epithelium* *Fiber* *OK* *1.53E+06* * 445558* *-1.78097* *-355.367* * 0.00715* *0.016986* *yes* *ENSMUSG00000093098* *ENSMUSG00000093098* * Gm22641* *7:130162450-133124354* *Epithelium* *Fiber* *OK* *87894.1* * 36474.7* *-1.26887* *-0.59863* *0.4913* *0.587174* *no* * ENSMUSG00000089855* *ENSMUSG00000089855* *Gm15662* *10:105187662-105583874* *Epithelium* *Fiber* *OK* *42868.9* * 21566.5* *-0.99114* *-20.7066* *0.0186 * *0.039568* *yes* *ENSMUSG00000092984* *ENSMUSG00000092984* *Mir5115* * 2:73012853-73012927* *Epithelium* *Fiber* *OK* *21104.8* * 8317.49* * -1.34335* *-447.314* *0.0001* *0.000354* *yes* *ENSMUSG00000086324* * ENSMUSG00000086324* *Gm15564* *16:35926510-36037131* *Epithelium* *Fiber* * OK* *6443.35* * 3664.15* *-0.81433* *-1.52095* *0.2129* *0.301429* *no* * ENSMUSG00000092981* *ENSMUSG00000092981* *Mir5125* *17:23803186-23824739* * Epithelium* *Fiber* *OK* *5974.14* *2390.75* *-1.32127* *-0.34111* *0.5746* *0.661937* *no* I checked some forums and they said that this is the drawback of TopHat/Cufflink/Cuffdiff when dealing with short genes. But I am still not so clear about this. Anyone got the same problem? What can I do with this situation? Anyone suggests any other good tools to test for (1) differential gene expression OR (2) both differential gene expression and gene discovery? Thank you Thanh

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Problem with FTP
by GANDRILLON OLIVIER 18 Jul '13

18 Jul '13

Hi I am trying to connect though FTP to transfer large files. Up to today everything went fine. But I tried many times to connect today and each time the ftp connection was instantly interrupted. Thank's for your help. Best Olivier

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Amplicon Sequencing Questions
by Seth Stern 18 Jul '13

18 Jul '13

Hi Everyone, I've got an amplicon that has fixed and variable sequence segments, and Im sequencing it with 180 base MiSeq reads. I'd like to align the reads using the fixed segment. Is this an appropriate job for either Bowtie or BWA (using a reference sequence that is necessarily shorter than the reads) ? If not, how should I do it ? Thanks, Seth Seth Stern, Ph.D. sethster(a)comcast.net 650 996 8726 (cell)

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database associated with galaxy server
by Jennifer Jackson 18 Jul '13

18 Jul '13

Hello Mitali, There is a rice genome available for use with most tools already on the public Main Galaxy server at https://main.g2.bx.psu.edu (http://usegalaxy.org) On the tool form, for the option to select a reference genome, or on the Upload or Edit Attributes form when selecting genome/build, type in the keyword "rice" to bring up: Rice (Oryza sativa): oryza_sativa_japanica_nipponbare_IRGSP4.0 For genomes not included as built-in native indexes (perhaps you wish to use a different strain), a Custom reference genome can be also used with most tools on Main. Instructions are here: http://wiki.galaxyproject.org/Support#Custom_reference_genome There are a few tools on Main that do not allow for Custom reference genomes - these have a fixed set of specified target genomes. Megablast is one such tool. To use an alternate genome with this tool and the few others like it, a local or cloud Galaxy is required. Instructions are here: http://getgalaxy.org http://usegalaxy.org/cloud If you need to add a reference genome to a local Galaxy (a cloud Galaxy would already include everything currently on Main), you can create the indexes yourself, or rsync our files for use in your instance. The rsync server also lists all genomes currently indexed on Main. Instructions are here: http://wiki.galaxyproject.org/Admin/Data%20Integration http://wiki.galaxyproject.org/Admin/NGS%20Local%20Setup Hopefully this helps, Jen Galaxy team http://wiki.galaxyproject.org/Support#Mailing_Lists On 7/17/13 11:43 PM, Mitali Merchant wrote: > hi, > > i wanted to know the genome databases associated with Galaxy server. i > want to perform peak calling using galxy server of the data of Oryza > Sativa (rice genome) but since this genome is not available in galaxy, > i want to know how can i get this genome as reference. > Kindly reply as soon as possible,it will be of great help > > Mitali Merchant > IIIT,Hyderabad > India -- Jennifer Hillman-Jackson Galaxy Support and Training http://galaxyproject.org

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