Differential Expression analysis workflow

  • Anna


    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/
    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,

    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 &

    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

    (Cross posted to SeqAnswers)

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