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  • ScottC
    Senior Member
    • Jan 2008
    • 244

    #1

    Pooling multiplexed libraries after ligation, before PCR

    Does anyone do this with the TruSeq kits? Illumina advertised this feature very strongly in their presentations in my area, as a huge time saving feature of the TruSeq kits as compared to the older sample prep kits... yet the product manual doesn't say anything about it. I'm a bit worried about uneven amplification of the libraries during a pooled PCR. Any comments?
  • chenhongli
    Junior Member
    • Jan 2011
    • 1

    #2
    Would that also cause jumping errors and waste of sequencing reads?

    Comment

    • jluk
      Junior Member
      • Dec 2015
      • 2

      #3
      Hi I'd like to refresh this thread. Has anyone had success with TruSeq library prep, if you pool your adapter ligated samples prior to the PCR enrichment step?

      Comment

      • HESmith
        Senior Member
        • Oct 2009
        • 512

        #4
        Pooling prior to PCR amplification is not recommended. It precludes the option of adjusting the concentrations of individual libraries to obtain equal read depth.

        Comment

        • Ancestro
          Member
          • Oct 2015
          • 15

          #5
          Done once. DO NOT attempt it. Some libraries just dissapeared.

          Comment

          • LOH
            Registered Vendor
            • Jul 2010
            • 23

            #6
            We didn't do that. In theory, it may cause unbalanced library pool. Some libraries could have very low sequencing reads.

            Comment

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