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  • Key Pass failure in one quad -- amplicon

    We have recently upgraded to the FLX+. We have had some successful quads with amplicons in the 600 to 800 base range. However, one quad with 16 samples shows nearly 100% key pass failure! It had a 20% enrichment similar to the quad beside it that was OK. The same barcode adaptors were used. The amplicons were amplified several cycles longer. By eye it is possible it might have more beads loaded than the other quad. Does anyone know what factors can cause such a large number of wells to have key pass failures?The control beads are proportionally low as well.

  • #2
    Are you using kits XLR70 ou XL+?

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    • #3
      XL+. The other quads were fine. Transcriptomes in two and amplicon similar to this one in the 3rd.

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      • #4
        Can you look through the PTP images for each of the early cycles by eye using gsRunBrowser to see if the key bases are there? Most sample keys are not compatible -- so if the library somehow got a little of a different sample key mixed in, it might cause issues.

        --
        Phillip

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        • #5
          We had a breakthrough on this Friday afternoon. Although we thought it was a problem with our samples in that Quad at first, after support looked at it, they were certain it was not analyzed properly. I had mentioned some error messages I had had when discussing this problem earlier but they didnt think it was the problem. NOW it probably is! I am thinking that this FLX+ data could be overwhelming the processing of the data. I guess the biggest hint was poor control beads as well.

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