Hello Yao,

The Ensembl-sourced reference annotation can often work with Cufflinks, however it does need to be in GTF format (the file samples listed here are not in GTF format). Also, you will need to alter the chromosome names once loaded into Galaxy. Specifically, Ensembl names chromosomes for human as "1", "2", "3", etc. and to have them match exactly with the Galaxy cashed human reference genome a "chr" needs to be added to create "chr1", "chr2", "chr3". A workflow to do this transformation is on the FAQ wiki here: http://main.g2.bx.psu.edu/u/jeremy/p/transcriptome-analysis-faq#faq5


Other issues with Ensembl GTF files have been known to pop up, so these data are not fully supported and we still do recommended using UCSC despite the missing gene_id information. But if you want to try, there is likely some sort of work-around that you could create on your own should a problem come up.

Hopefully this helps,

Jen

On 8/2/11 6:30 PM, yao chen wrote:
Dear all,

I have a similar problem when using cufflinks in galaxy (net version).
If I didn't select the reference annotation, I can get the FPKM
values,but since no reference,I can not get the transcript or gene name.
It looks like these:

test_id gene_id gene locus sample_1 sample_2 status value_1 value_2
ln(fold_change) test_stat p_value q_value significant
TCONS_00000002 XLOC_000025 - chr1:33860011-33860048 q1 q2 NOTEST
1.794e+06 0 -1.79769e+308 -1.79769e+308 0.0188163 1 no

There is no gene_id. However, if I use the reference annotation
downloaded from ENSEMBLE.I can get the gene_ids, but there FPKM values
are all "0":

tracking_id class_code nearest_ref_id gene_id gene_short_name tss_id
locus length coverage status FPKM FPKM_conf_lo FPKM_conf_hi
ENSMUSG00000024232 - - ENSMUSG00000024232 Bambi - 18:3507954-3516402 - -
OK 0 0 0
ENSMUSG00000091539 - - ENSMUSG00000091539 Vmn1r238 - 18:3122454-3123465
- - OK 0 0 0

------------------
Any thoughts?

2011/8/3 Jennifer Jackson <j...@bx.psu.edu <mailto:j...@bx.psu.edu>>

    Hi Aleks,

    Thanks for sending the data link, this helped to narrow down the
    root cause of the issue.

    The UCSC-sourced GTF file has the attributes gene_id and
    transcript_id set to the same value (both as transcript_id). The
    result of this is that each transcript is interpreted by Cufflinks
    as a single gene, with no gene grouping (thus no isoforms).

    We have plans to develop a work-around. This would likely involve
    (for the refGene track in particular) the value in the UCSC's
    primary table refGene.name2 being swapped into the refGene GTF
    file's gene_id value. This would generate accurate gene-level
    statistics when the file is used as input to Cufflinks. You could do
    the same swap (outside of Galaxy) if you wanted to give it a try and
    have resource.

    Very sorry for the current inconvenience,

    Best,

    Jen
    Galaxy team

    On 7/25/11 11:26 AM, Jennifer Jackson wrote:

        Hello Aleks,

        Chromosome names must be exact between all input files). Also,
        the SAM
        file and GTF file both must be sorted the same way. This FAQ may
        be of
        interest:
        http://main.g2.bx.psu.edu/u/__jeremy/p/transcriptome-__analysis-faq
        <http://main.g2.bx.psu.edu/u/jeremy/p/transcriptome-analysis-faq>

        If still a problem, please share the history with me directly either
        using my email address or generate the share link and email to me
        (only). Use "Options -> Share or Publish", not just your sessions
        browser URL.

        Best,

        Jen
        Galaxy team

        On 7/22/11 8:45 AM, Aleks Schein wrote:


            Dear all,
            I am trying to run Cufflinks installation in Galaxy on
            Solexa RNAseq
            samples from HeLa cells.
            Running Cuffcompare, according to the manual, should produce
            a tmap
            file, listing FMI values for detected isoforms. However, my
            files only
            have either "100" or "0" in FMI field. And FPKM column
            contains only
            zeros.
            Is there something wrong with my input files, or parameter
            settings? Or
            is it rather a specific issue with Galaxy Cufflink's
            installation?

            The data in question is available here:
            http://main.g2.bx.psu.edu/u/__aleks/h/guided-__assemblyadvanced
            <http://main.g2.bx.psu.edu/u/aleks/h/guided-assemblyadvanced>

            Thanks,

            Aleks Schein



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