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  • Pair-end Key Sequence

    Hi everyone!

    I am currently testing pair-end library on my GS Jr and I was wondering if I can multiplex one PE library with shotgun ones?

    This thread http://seqanswers.com/forums/showthr...t=pair-end+454 says no but it was almost 2 years ago.

    I asked my "Roche guy" and he told me that the key sequence (GACT) was the same but I want to be sure before starting the experiment...
    Last edited by LuLuNe; 01-08-2013, 03:11 AM.

  • #2
    No, do not mix them. Paired-end aka amplicon A kit (LibA) uses tcag key. Would you have used a General Library protocol for the shotgun then both would be using tcag key.

    But, still, I bet the software will not identify your mate-pairs.

    And, I am not talking about control beads. The software is obviously not able to cope with all kinds of crazy combinations of adapters, MIDs, linkers, keys, control sequences, etc. Bear in mind it is also computationally much more demanding to test all such combinations. In brief, they are likely not tested all together, and won't, ever. Use the rubber gasket to split the PTP device into multiple regions ... (ah, GS Junior). Then I am sorry.

    The gact key you mention appears only in RapidLib-based shotgun datasets, not in ampliconA/paired-end data.
    Last edited by martin2; 02-11-2013, 07:11 PM.

    Comment


    • #3
      Hi there. The new Paired End Rapid Library Protocol uses the same RL adaptors, or RLMID adaptors, and Lib-L chemistry as the shotgun Rapid Libraries. Thus they'll have the same GACT key as your "Roche guy" said. If you used RLMID adaptors for your PE library and the shotgun one, they could be demultiplexed after. But the thing to think about are the emPCR conditions. I think they are different between PE libraries and shotgun libraries which would make multiplexing them not really appropriate.

      Lib-A chemistry is only for bidirectional amplicon sequencing, which is not really paired end.

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