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  • Indexed libraries best pooling strategy

    Hi
    We recently sequenced 20 indexed libraries on one lane of Hi-Seq. Unfortunately, the pooling strategy we used based on bioanalyzer data was not even.

    Any suggestion on the best pooling strategy to get uniform pool?

    Dinesh

  • #2
    Quantitate your libraries by qPCR, not BioA.

    Comment


    • #3
      Search the forum for threads addressing accurate library quantification. There are advantages and limits to each technique. For example, adapter dimer contamination can wreak havoc with qPCR measurements.

      Harold

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      • #4
        Hello spdinesh,
        In our experience, it is best to use multiple quantification platforms and go from there (bioanalyzer, Qbit, nanodrop), while knowing that +/- 20% is expected from pooling. Even pipette error can make a difference when pooling at n=20 with dilutions.

        What was the standard dev. seen from your libraries?

        jon

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