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  • questions about RNA-Seq

    Hey guys, I am a little confused about RNA-Seq. Today I mapped the reads in a RNA-Seq expriment onto the genome, and I found that for one gene locus, the read can map to both the forward strand and the reverse complement strand. I am not sure this is the right result since I am familar with the expriment, I hope you can help me to figure this out.

    Given a specific mRNA, the RNA-Seq result will give many short reads. These reads can map to the corresponding gene on the genome. I know that the mRNA will be converted to double-stranded cDNAs before sequencing. My question is that, if we map the read to the genome, is that possible that the reads coming from the same single strand mRNA can map to both strand of the genome? In another words, can the sequencing machine know which one of two strands of the cDNA need to be sequenced?

    If the machine randomly choose either strand to sequence, after we map the read to the genome. how can we define which strand of the genome is the mRNA template?

    Thank you very much

  • #2
    illumina's standard mRNA-seq is *not* strand specific.

    check out illumina's 'directional mRNA-seq' protocol -- that protocol preserve strand.

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    • #3
      I think the sequencer read either strand, because adaptors don't look at the orientaiton of DNA fragment during the adaptor ligation process.

      Strand specific RNA-Seq protocols may help you to solve the problem.
      High-throughput complementary DNA sequencing (RNA-Seq) is a powerful tool for whole-transcriptome analysis, supplying information about a transcript's expression level and structure. However, it is difficult to determine the polarity of transcripts, and therefore identify which strand is transcribed …

      I haven't tried this yet though.

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      • #4
        Thank you very much. I only want to make sure that standard RNA-Seq will map reads to both strand. but also thank you for your new information about the new technology.


        Originally posted by pzumbo View Post
        illumina's standard mRNA-seq is *not* strand specific.

        check out illumina's 'directional mRNA-seq' protocol -- that protocol preserve strand.
        Originally posted by kenosaki View Post
        I think the sequencer read either strand, because adaptors don't look at the orientaiton of DNA fragment during the adaptor ligation process.

        Strand specific RNA-Seq protocols may help you to solve the problem.
        High-throughput complementary DNA sequencing (RNA-Seq) is a powerful tool for whole-transcriptome analysis, supplying information about a transcript's expression level and structure. However, it is difficult to determine the polarity of transcripts, and therefore identify which strand is transcribed …

        I haven't tried this yet though.
        Last edited by found; 07-11-2010, 08:36 PM.

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