[galaxy-user] NGS: Indel Analysis tool order
Hello! I have a question about the NGS: Indel Analysis toolset in Galaxy. I have aligned my samples from Illumina's HiSeq2000 to the reference genome using BWA. I've called SNPs using SAMTools and now need to call indels. Under the NGS: Indel Analysis toolset, I see two options: Filter Indels for SAM and Extract indels from SAM. Since the descriptions are a little vague, I would presume that I start with the Filter Indels for SAM first on the BWA SAM file and then run Extract indels from SAM on the Filter output SAM file (i.e. BWA SAM - Filter Indels for SAM - Extract indels from SAM). Is this the correct order? Or would I skip the Filter Indels for SAM step (since the BWA SAM file technically already contains indels so there would be no need to filter) and just go straight to Extract indels from SAM (i.e. BWA SAM - Extract indels from SAM)? I've tried both ways and get different results. For example: 1. BWA SAM - Filter Indels for SAM - Extract indels from SAM - gives me 26,417 regions of interest 2. BWA SAM - Extract indels from SAM - gives me 93,974 regions of interest Which way is correct? Any help/info would be greatly appreciated! Thanks, David ___ 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 question about using an unspecified (?) database/build
Hello! I have an RNA-Seq project which consists of 5 samples from the species tree shrew. When uploading these fastq files into Galaxy, I chose unspecified (?) for the database/build since the latest tree shrew version is not in the drop down list. When using TopHat, Cufflinks/Compare I have selected a reference genome from my history instead of using a built-in index, as well as a gtf annotation file for Cufflinks/Compare and everything has been working fine. Now, I am at the Cuffdiff step and I am running into an error when setting it up to perform replicate analysis. When I select my TopHat accepted hits bam file I see a red X and the error: Unspecified genome build, click the pencil icon in the history item to set the genome build. Here's a screenshot of what I'm seeing: [cid:image001.png@01CC5E4E.76F37AF0] Since the latest reference genome for tree shrew wasn't listed, that's why I chose unspecified (?). Should I go back and edit these accepted hits bam files to say the Database/Build from the drop down list is Tree shrew Dec. 2006 (Broad/tupBel1) (tupBel1)? I know that this is simple to change, but will this affect my results in any way? Any help/info would be greatly appreciated. Thanks, David inline: image001.png___ 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] Cuffdiff question about using an unspecified (?) database/build
Jen, Thank you very much for the reply. I'm glad to know it is a known bug and not something on my side of things. So, would my analysis be affected if I did change the bam file Database/Build to the older tree shrew version found in the drop down list? What significance does this Database/Build box have in downstream analysis if you have your own fasta reference genome file and gtf annotation file that is being referenced instead of a locally cached one? I'm just trying to obtain a better understanding of the Database/Build box for analyses where I provide the fasta and gtf file. Thanks, David -Original Message- From: Jennifer Jackson [mailto:j...@bx.psu.edu] Sent: Friday, August 19, 2011 9:20 AM To: David K Crossman Cc: galaxy-user (galaxy-user@lists.bx.psu.edu) Subject: Re: [galaxy-user] Cuffdiff question about using an unspecified (?) database/build Hello David, This is a known bug. The correction is planned to be moved out onto the public Galaxy instance at the next update (within a week). Sorry for the current inconvenience, Best, Jen Galaxy team On 8/19/11 7:00 AM, David K Crossman wrote: Hello! I have an RNA-Seq project which consists of 5 samples from the species tree shrew. When uploading these fastq files into Galaxy, I chose unspecified (?) for the database/build since the latest tree shrew version is not in the drop down list. When using TopHat, Cufflinks/Compare I have selected a reference genome from my history instead of using a built-in index, as well as a gtf annotation file for Cufflinks/Compare and everything has been working fine. Now, I am at the Cuffdiff step and I am running into an error when setting it up to perform replicate analysis. When I select my TopHat accepted hits bam file I see a red X and the error: Unspecified genome build, click the pencil icon in the history item to set the genome build. Here's a screenshot of what I'm seeing: Since the latest reference genome for tree shrew wasn't listed, that's why I chose unspecified (?). Should I go back and edit these accepted hits bam files to say the Database/Build from the drop down list is Tree shrew Dec. 2006 (Broad/tupBel1) (tupBel1)? I know that this is simple to change, but will this affect my results in any way? Any help/info would be greatly appreciated. Thanks, David ___ 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/Support ___ 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 files produced by Barcode Splitter
Jeremy, The files need to be groomed using the FastQ Groomer so that they will end up in the fastqsanger state. Then your files will show up in the pull-down menus. David From: galaxy-user-boun...@lists.bx.psu.edu [mailto:galaxy-user-boun...@lists.bx.psu.edu] On Behalf Of Jeremy Coate Sent: Monday, July 18, 2011 1:44 PM To: galaxy-user@lists.bx.psu.edu Subject: [galaxy-user] using files produced by Barcode Splitter I used the Barcode Splitter tool to split multiplexed RNA-Seq libraries into separate files. I would now like to map the reads from each of these fastq files to a reference genome. However, the fastq files generated by Barcode Splitter don't appear in the Fastq File pull-down menus within the the BWA or Bowtie launch pages. I'm probably missing something obvious, but what is the trick for making these files available for the mapping tools? Do I need to import them into my history somehow? Thanks! Jeremy ___ 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] Mycoplasma pneumoniae M129 and FH reference genome
Hello! I noticed that Mycoplasma pneumonia M129 and FH are not found in the reference genome in Galaxy. Would it be possible to have both of those in there? Thanks, David ___ 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] RNA seq analysis and GTF files
Jeremy, Thank you very much for this information. One quick question. I added the gene_id values to the 10th column of my patched GTF file. After uploading it to Galaxy, the column doesn't have a name (i.e. column 1 = Seqname; column 2 = Source; etc...). Do I need to assign it a name (i.e. gene_name or gene_id) for it to be recognized and if so, how do you assign column names to GTF files? Thanks, David From: Jeremy Goecks [mailto:jeremy.goe...@emory.edu] Sent: Thursday, April 07, 2011 9:40 PM To: David K Crossman Cc: galaxy-user Subject: Re: [galaxy-user] RNA seq analysis and GTF files David, Your analysis looks reasonable. In fact, in your isoform tracking FPKM file you get nearest_ref_id, so that's promising. What I think is needed is the addition of an attribute called gene_name to your reference file; you can use whatever value you want for gene name, and using the same value as gene_id probably makes sense. Rerun your analysis with the further-patched GTF file, and let us know if this doesn't solve the problem. Also note that even using this attribute, some gene name/ids and some nearest_ref_id columns will not be populated in some cuffdiff files. See the post from Howie in this thread for an explanation from a Cufflinks developer: http://seqanswers.com/forums/showthread.php?t=6288 Best, J. On Apr 7, 2011, at 5:00 PM, David K Crossman wrote: Jeremy, I've shared it with you using your email address. Thanks, David From: Jeremy Goecks [mailto:jeremy.goe...@emory.edu] Sent: Thursday, April 07, 2011 3:42 PM To: David K Crossman Cc: galaxy-user Subject: Re: [galaxy-user] RNA seq analysis and GTF files David, can you please share your history with me and I'll take a look (History Options -- Share/Publish -- Share with User -- my email? Thanks, J. On Apr 7, 2011, at 3:23 PM, David K Crossman wrote: Hello! I would like to ask a question related to this thread below. I ran into the same issues as below and was unaware of having to swap some columns around in the GTF file. So, after 'swapping the gene name from the complete table (name2 value, column 12) into the GFT file's gene_id value (which by default is the same as transcript_id), I uploaded this patched file (mm9) into Galaxy and ran Cufflinks, CuffCompare and CuffDiff using this patched GTF file as the reference annotation. For both Cufflinks and CuffCompare, the gene_id was present in their respective columns. The problem I have encountered now is that in all of the output files in CuffDiff, the gene_id column is blank (contains a -; highlighted in yellow below). This example is from the CuffDiff gene expression output file: test_id gene locus sample_1 sample_2 status value_1 value_2 ln(fold_change) test_stat p_value significant XLOC_01 - chr1:4797973-4836816 q1 q2 OK 73.1908 82.1567 0.115559 -0.71896 0.472168 no XLOC_02 - chr1:4847774-4887990 q1 q2 OK 81.7264 53.1165 -0.43089 2.44474 0.014496 no XLOC_03 - chr1:5073253-5152630 q1 q2 OK 408.289 333.749 -0.20159 2.73173 0.0063 no XLOC_04 - chr1:5578573-5596214 q1 q2 NOTEST 2.34764 4.79772 0.71473 -0.89735 0.369532 no What am I doing wrong? I am interested in the differentially expressed genes in this RNA-Seq dataset (as well as calling variants, which is my next step, but want to get this answered first before moving on). Any info, suggestions or help would be greatly appreciated. Thanks, David -Original Message- From: galaxy-user-boun...@lists.bx.psu.edumailto:galaxy-user-boun...@lists.bx.psu.edu [mailto:galaxy-user-boun...@lists.bx.psu.edu] On Behalf Of Jeremy Goecks Sent: Friday, April 01, 2011 8:47 AM To: ssa...@ccib.mgh.harvard.edumailto:ssa...@ccib.mgh.harvard.edu Cc: galaxy-user Subject: Re: [galaxy-user] RNA seq analysis and GTF files On Mar 31, 2011, at 12:30 PM, ssa...@ccib.mgh.harvard.edumailto:ssa...@ccib.mgh.harvard.edu ssa...@ccib.mgh.harvard.edumailto:ssa...@ccib.mgh.harvard.edu wrote: Hi Jeremy, I used your exercise to perform an RNA-seq analysis. First I encountered a problem where the gene IDs were missing from the results. Jen from the Galaxy team suggested this: Yes, the team has taken a look and there are a few things going on. The first is that when running the Cuffcompare program, a reference annotation file in GTF format should be used in order to obtain the same results as in Jeremy's exercise. This seemed to be missing from your runs, which resulted in badly formatted output that later resulted in a poor result when Cuffdiff was used. The second has to do with the reference GTF file itself. For the best results, the GTF file must have the gene_id attribute defined in the 9th column of the file and the chromosome names must be in the same format as the genome native to Galaxy. Depending on the source of the reference GTF, one
