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  • is ok to analyse 21 bp DGE tags with tophat-cufflink pipeline?

    Hi friends,

    I have 21 bp illumina RNA-seq tags samples across multiple tissues, and the reference genome sequence.

    Is it Ok to use Tophat(which implement bowtie) and cufflink pipeline (http://www.nature.com/nprot/journal/....2012.016.html) to do differential expression analysis?

  • #2
    It is pointless. You will not be able to map any reads across splice junctions. Use Bowtie instead.

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    • #3
      Originally posted by Dario1984 View Post
      It is pointless. You will not be able to map any reads across splice junctions. Use Bowtie instead.
      Thanks! So after I generated SAM files of multiple samples with "Bowtie", I still could use "cuffdiff" (in cufflink) for differential expression analysis with the SAM files, gene annotation(coordinate file, gtf) and the genome sequence (genome_assembly.fasta)?

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      • #4
        Tophat actually maps to the transcriptome first, so you would still get exon-exon junctions, even with these very short reads (though you're unlikely to discover new ones, if that's important to you). So yes, you could use the normal tophat->cufflinks->cuffdiff pipeline with these sorts of reads.

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        • #5
          Originally posted by dpryan View Post
          Tophat actually maps to the transcriptome first, so you would still get exon-exon junctions, even with these very short reads (though you're unlikely to discover new ones, if that's important to you). So yes, you could use the normal tophat->cufflinks->cuffdiff pipeline with these sorts of reads.
          Thanks for your suggestion. I was thinking the same way. Gene structure/alternative splicing is not important to me at the moment. I just want to measure expression variation of loci among samples(different tissues).

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