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-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.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.edu<mailto:ssa...@ccib.mgh.harvard.edu>>
<ssa...@ccib.mgh.harvard.edu<mailto: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 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.
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___________________________________________________________
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:
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