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  • Strand specific tophat/cufflinks

    Dear all,

    I have a strand-specific RNA-seq library to assemble (Illumina). I would like to use TopHat/Cufflinks. From the manual of TopHat, it says,

    "--library-type TopHat will treat the reads as strand specific. Every read alignment will have an XS attribute tag. Consider supplying library type options below to select the correct RNA-seq protocol."

    Does it mean that TopHat only supports strand-specific protocols? I use option "--library-type fr-unstranded" to run, does it mean it runs in a strand-specific way? I googled it and asked the developers, but got no answer...

    I got some result:
    Here the contig is assembled by two groups of reads, left side are reverse reads, while right side is forward. (for visualization, i have reverse complement the right mate)

    But some of the contigs are assembled purely from reverse or forward reads. If it is strand specific, one gene should produce the reads in the same direction. It should not report the result like the image above, am I right? Or is it possible that one gene is fragmented and then sequence independently, so that happenly left part produce reverse reads while right part produce forward reads? From my understanding, the strand specificity is kept by 3'/5' ligation, so should be in the unit of genes.

    What is the problem here? Or did I understand the concept of 'strand specific' wrongly? Any help is appreciated.

  • #2
    Are these Illumina paired reads? You would expect those to come in a forward/reverse pair (even if the RNA was strand specific).

    Comment


    • #3
      Thans, maubp.

      Yes, they are Illumila. When assemblying, I passed them in as FR format. I just double checked with that.

      I reverse complement the right mates and aligned back to the contigs. Such that I have a better idea about their directions.

      I have got some contigs assembled all by forward (or reverse) reads. Meanwhile, I got some contigs shown as the image above. So I asked here.

      Comment


      • #4
        Is that image typical, or an odd case? Are most of your 'genes' supported by strand specific reads (after the processing you described).

        Perhaps the gene shown is a miss-assembly, and is really two genes on opposite strand which have been joined at the 3' end. If so, you may be able to check that with BLAST or domain searches.

        Comment


        • #5
          So they may be from different genes? Just wrongly joined together? I would check it. Thanks.

          Comment

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