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.goecks@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_000001

chr1:4797973-4836816 q1 q2 OK 73.1908 82.1567 0.115559 -0.71896 0.472168 no XLOC_000002

chr1:4847774-4887990 q1 q2 OK 81.7264 53.1165 -0.43089 2.44474 0.014496 no XLOC_000003

chr1:5073253-5152630 q1 q2 OK 408.289 333.749 -0.20159 2.73173 0.0063 no XLOC_000004

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-bounces@lists.bx.psu.edu [mailto:galaxy-user-bounces@lists.bx.psu.edu] On Behalf Of Jeremy Goecks Sent: Friday, April 01, 2011 8:47 AM To: ssassi@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, ssassi@CCIB.MGH.HARVARD.EDU ssassi@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 of these may need to be adjusted. Chromosome names can be adjusted using Galaxy's "Text Manipulation" tools. The gene_id attribute would need to be adjusted prior to loading into Galaxy.

For mm9, using the "Get Data -> UCSC Main table browser" tool can help you to obtain all of the raw data necessary to create a complete GTF file with a gene_id identifier. Extract data from the track "RefSeq Genes" and output the primary data table "refGene" twice - first in GTF format, then again as the complete table in tabular format (not BED). Then, using your own tools, swap in the gene name from the complete table (name2 value, column 12) into the GTF file's gene_id value (which by default is the same as transcript_id). Upload and the tools will function as intended.

The team is aware of the issues associated with GTF source files and is discussing solutions. Any changes to native data content will be reported to the mailing list in a News Brief or other communications.

Our apologies for the inconvenience! Thanks for using Galaxy and please let us know if we can help again,

Best,

Jen Galaxy team"

I followed the directions (or at least I think I did) and things seemed to work better but there is one more issue for example in file: Galaxy287-[Cuffdiff_on_data_197,_data_197,_and_data_274__isoform_FPKM_ tracking].tabular.txt The column gene_short_name does not have any names in it. nearest_ref_id does have the gene ID info so I can still interpret the data, but I was wondering if there remains another problem that I'm not aware of with the GTF file.

Slim,

Please send questions to the galaxy-user mailing list (cc'd) rather than individual Galaxy team members; there are many people on the list that may be able to address your question, and discussions are archived for future use as well. Without seeing your analysis, I'd suggest trying two things:

(1) Provide gene annotation reference file to Cufflinks as well as Cuffcompare and Cuffdiff; in other words, you'll want to do guided assembly. (2) Try using an Ensembl GTF, which has the gene name in the attributes.

I think (2) is more likely to generate the results you want, but there are the many known problems in using Ensembl GTFs with Cufflinks/compare/diff.

Good luck, J. ___________________________________________________________ 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:

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