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  • % of reads mapped to transcriptome

    Hello,

    I am doing RNA-Seq analysis and needed some advice on mapping PE reads back to the reference transcriptome. I made the reference from the set of 12 RNA-Seq reads (after duplicates) were removed.

    I am then mapping each RNA-Seq dataset back to the transcriptome, but I am only getting about 60% mapping back to the reference transcriptome. Is it possible that my fragment length is too short, thus precluding reads to be mapped back to my reference? I have the mean size of the fragments from the sequencing facility and I've set the program (CLC GW) to accomodate a range 75 bp below and above the mean size (fragment length between 150 and 300).

    Or should I start looking to relax some of the parameters that influence the matching of reads to the reference?

    Thanks,
    Andor

  • #2
    Andor,

    You should consider the quality of your transcriptome assembly as, in my experience, CLC will very often do a poor job of reconstruction. For instance, how many full length transcripts do you have? How many match to transcripts from the closest reference using a reciprocal best blast procedure?

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    • #3
      Your reference is basically singletons or contigs from assembly of non-redundant reads from all libraries. So, all of them should map back to the reference.

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      • #4
        Originally posted by peromhc View Post
        Andor,

        You should consider the quality of your transcriptome assembly as, in my experience, CLC will very often do a poor job of reconstruction. For instance, how many full length transcripts do you have? How many match to transcripts from the closest reference using a reciprocal best blast procedure?
        So I did an assembly on CLC using 454 data and it was very good - 85% got a blastx hit. But this illumina data, not so much. Might try a different assembler...

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        • #5
          Originally posted by nucacidhunter View Post
          Your reference is basically singletons or contigs from assembly of non-redundant reads from all libraries. So, all of them should map back to the reference.
          right...theoretically...or at least a VERY high percentage...

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