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  • 454 amplicon recurring problem - wide, large untrimmed distribution

    Hello,
    We have done several amplicon sequencing projects lately that have yielded poor results. Link below is to a summary of thoses runs including Bioanalyzer traces, untrimmed and trimmed distributions for several of them (pword is "amplicon"):



    Good runs were on:

    April 22
    June 7
    July 31
    Oct 19 (Region 2)

    Then the weird distributions started (wide and too-high untrimmed, dismal trimmed):

    Nov 3
    Nov 7
    Nov 18 (partial)
    Nov 26 (partial)

    We are upgraded to GSFLX-XL+, but are still using regular Titanium kits.

  • #2
    Hi Indiana,
    When was your instrument upgraded to XL+?
    Any issues with your shotgun runs?

    What parameters are you using for signal processing? (You would probably have to get that info from your IT guys.)

    --
    Phillip

    Comment


    • #3
      I am interested to know if there is some issue with your instrument running Titanium since you got the upgrade or, as I suspect, the runs are fine but the way your IT guys tuned your amplicon processing pipeline is not compatible with Titanium-run-on-a-FLX+ data.

      --
      Phillip

      Comment


      • #4
        Hi everybody, I just performed an amplicon run to sequence 16S RNA using fusion primers and the "one way read approach". The amplicons have ~700bp and the bioanalyzer profiles were perfect with no primer-dimer peak. Our machine was upgraded to GS FLX+ but we used XLR70 reagents. Guess what? I obtained a high % of short read/short primer, reads with low quality. The processing was made using amplicon and shotgun pipelines giving the same poor result. Amplicon sequencing using 454 shouldn't be allowed. I would appreciate any advise from you. Thanks in advance.

        Comment


        • #5
          LFMar, what emPCR conditions did you use?

          Comment


          • #6
            Originally posted by RCJK View Post
            LFMar, what emPCR conditions did you use?
            I used standard emPCR conditions

            Comment


            • #7
              Try using the emPCR conditions for long amplicons and see if that helps.

              Comment


              • #8
                Originally posted by LFMar View Post
                Hi everybody, I just performed an amplicon run to sequence 16S RNA using fusion primers and the "one way read approach". The amplicons have ~700bp and the bioanalyzer profiles were perfect with no primer-dimer peak.
                ssDNA primer-dimers can anneal to the adapters of your 700 bp amplicons and effectively "hide" from size/molecular weight-based fractionation methods. I was hearing that people were getting better results on PCR products (which is what fusion amplicons are) when they were subjected to 2 cycles of AmPureXP clean-up.

                AmPure is a size-based fractionation method, but perhaps by doing 2 cycles the primer-dimer strands exchange off the full length amplicons enough to get a good result.

                Or, you could go old-school and run a denaturing acrylamide gel. That way all the strands run separately and the primer-dimers can't hide.

                --
                Phillip

                Comment

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