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  • 3' bias

    What is 3' bias exactly? I understand it is something caused by the enrichment of poly(A) mRNA using oligo(dT) primer.(or is that wrong?) How does it cause the 3' bias?

  • #2
    See this previous SeqAnswers thread: http://seqanswers.com/forums/showthread.php?t=9839

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    • #3
      The thread does not explain what 3' bias is.

      Comment


      • #4
        Since polyA tails are at the 3'-end of mRNA's any method that uses polyA sequence for enrichment is likely to recover fragments from that end.

        This leads to a greater chance of having sequences from the 3'-end in the resulting reads as opposed to reads that represent the 5'-end of the molecules.


        Originally posted by numfar View Post
        The thread does not explain what 3' bias is.

        Comment


        • #5
          So the mRNA has been fragmented? Is this intentionally?(if so why?)

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          • #6
            Originally posted by numfar View Post
            So the mRNA has been fragmented? Is this intentionally?(if so why?)
            Next generation sequencers, specifically those used for RNA-Seq experiments, require library fragments around 3-400 bases. However, RNA needs to be of good quality (non fragmented) at the beginning for successful mRNA enrichment (or rRNA removal)

            Typical RNA-Seq workflow goes a bit like this (based on Illumina TruSeq method):

            Enrich mRNA (usually PolyA enrichment by Oligo dT beads x2)
            Fragment mRNA to required size (usually metal ion-catalyzed base hydrolysis fragmentation)
            Reverse transcribe to make double stranded cDNA
            Library prep (end repair, adenylate, ligate adapters, size select, PCR enrich)
            QC and Quantify library
            Sequence

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