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  • Differential expression analysis workflow in Cufflinks

    Hi,

    I'm hoping that someone can help me, as I couldn't work out how to do
    this from the manual. Would someone be able to give me the steps in a
    differential expression analysis?

    I have run tophat with the following command for each of my two solexa
    sequence.txt RNA-seq files seperately:

    tophat --solexa1.3-quals -p 2 -o 101/100315/tophat/
    ~/software/bowtie-0.12.2/indexes/m_musculus_ncbi37
    101/100315/s_2_sequence.txt &> 101/100315/tophat/tophat.out &

    I would like to get the expression levels for all Ensembl transcripts.
    I have downloaded this gtf file from Ensembl,
    ftp://ftp.ensembl.org/pub/current_gtf/mus_musculus,

    However when I run
    cuffdiff ~/data/gtf/Mus_musculus.NCBIM37.57.gtf
    101/100315/tophat/accepted_hits.sam 95/100315/tophat/accepted_hits.sam
    &> cuffdiff.out &

    or
    cufflinks -G ~/data/gtf/Mus_musculus.NCBIM37.57.gtf 101/100315/tophat/accepted_hits.sam &>
    101/100315/tophat/cufflinks.out &

    I get the following error

    Error: duplicate GFF ID 'ENSMUST00000127664' (or exons too far apart)!

    I'm pretty sure I've misunderstood the workflow, if someone could give me
    an overview of the steps and what gtf file I should be using that would
    be great.

    Many Thanks
    Anna

    (Cross posted to Bowtie forum)

  • #2
    I get the same error message, and if you look at the transcript ENSMUST00000127664 it is indeed very long and has an intron of size ~ 4.4 Mb. This is way above the default maximum intron length (300,000) and that is why you get this error.


    Also, make sure that your GTF file only contains rows for exons, not CDS or transcripts as well. Otherwise all your records in the GTF are duplicated. Maybe you have done this already, but just in case.
    Last edited by Boel; 04-27-2010, 06:35 AM. Reason: one more thing!

    Comment


    • #3
      Hi All,

      I met the same error with transcript ENSMUST00000127664, but if I use the Mus_musculus.NCBIM37.56.gtf and the relative index, I did not meet the error. Is that because some annotation has been updated?

      Well, I manually deleted the rows which contain transcript ENSMUST00000127664 in the annotation file, then the problem solved. I will try cuffdif later on. But what if people are interested in this gene/transcript? it might not be a good idea to delete? Hopefully someone can give good explanation!

      Thanks!
      Last edited by Wei-HD; 05-17-2010, 06:40 AM.

      Comment


      • #4
        Originally posted by Wei-HD View Post
        Hi All,

        I met the same error with transcript ENSMUST00000127664, but if I use the Mus_musculus.NCBIM37.56.gtf and the relative index, I did not meet the error. Is that because some annotation has been updated?

        Well, I manually deleted the rows which contain transcript ENSMUST00000127664 in the annotation file, then the problem solved. I will try cuffdif later on. But what if people are interested in this gene/transcript? it might not be a good idea to delete? Hopefully someone can give good explanation!

        Thanks!
        I met the same problem. Is this problem solved for you?

        Comment


        • #5
          Hi Gangcai,

          Sorry I did not figure out a solution about this, I just sticked to the old version index and GTF file, since all my samples were analyzed against the old index (NCBIM37.56). Also I use DESeq R package for all the gene expression level analysis.

          I wonder how other SEQers think?

          Thanks!

          Comment

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