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MiSeq Micro kit issue, no data except cluster density

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  • MiSeq Micro kit issue, no data except cluster density

    Hello everyone.

    Lurked for a while, but first time posting. I have a bit of a vexing issue.

    We are using a MiSeq Micro kit (v2 chemistry, 300 cycles).

    Libraries were mixed, denatured, PhiX added, loaded on the cartridge, etc.
    (15pM loading, 5% PhiX @ 10pM)

    The run started and it completed, but did not generate any data except cluster density (cluster density around 900, which I have already determined why it might be low).
    -no cluster diversity
    -no tile images
    -no % PF
    -no % aligned
    -no sequence data
    -no undetermined bins


    I don't believe it is a sample issue as the libraries were quantified using kapa, looked at via Tapestation, and re-quantified by Qbit for good measure (just to get library quality out of the way as a reason). and adjusting for a slight mistake calculating the library concentrations, the cluster density is what I would expect had the rest of the run work.

    PhiX had been previously denatured and used before and after this run with success.

    We have never used the Micro kit before and tech support had suggested there was nothing special you had to do or change in the sample sheet or LRM (since the kit's RFID code is read by the machine), but it is the only logical place that makes sense. This is based on the fact that the PhiX had worked on other runs that were using the Micro kit without issues, and for the % aligned to not show up with the PhiX makes me think there was a setting or something that made it so no data was collected.

    Has anyone ever seen an issue like this before? Could really use some help as I have been beating my head against my desk for the last 2 weeks.

  • #2
    Have you had Illumina tech support a look at this run? They can do that remotely. Sounds like there may be a hardware/software issue (assuming you have libraries that will amplify by qPCR).

    Comment


    • #3
      Originally posted by GenoMax View Post
      Have you had Illumina tech support a look at this run? They can do that remotely. Sounds like there may be a hardware/software issue (assuming you have libraries that will amplify by qPCR).
      We have worked with Illumina tech support. They say there is nothing wrong with the machine itself and said it is probably a bad library prep. But Illlumina loves saying that. It's always the library prep haha.

      However, I don't believe that it is. library showed up by qPCR, Qbit, and Tapestation, with the results we would expect.

      I am just wondering if anyone here has experience with the MiSeq Micro kit and if there is anything you have to do to run that kit vs the regular v2 or v3 kit.

      Comment


      • #4
        By "no % PF", do you mean, PF% was zero, or PF% was not calculated? If the latter, then that is an issue independent of library quality. I have never seen this, but it sounds more like an issue with the computer.
        The micro and nano cassettes are identical to the standard cassettes. Near as I can tell, Illumina literally blacks-out part of the flowcell to "create" a micro or nano kit.
        --
        Phillip

        Comment


        • #5
          Originally posted by pmiguel View Post
          By "no % PF", do you mean, PF% was zero, or PF% was not calculated? If the latter, then that is an issue independent of library quality. I have never seen this, but it sounds more like an issue with the computer.
          The micro and nano cassettes are identical to the standard cassettes. Near as I can tell, Illumina literally blacks-out part of the flowcell to "create" a micro or nano kit.
          --
          Phillip
          Hi Phillip,

          Thanks for the response! You are correct, there was no passing filter calculated. Same for alignment or cluster diversity, and no fastq data generated. Only the cluster density was generated.

          Comment


          • #6
            Originally posted by KyleWolf View Post
            Hi Phillip,

            Thanks for the response! You are correct, there was no passing filter calculated. Same for alignment or cluster diversity, and no fastq data generated. Only the cluster density was generated.
            Are you getting the cluster density from SAV? If not, where?

            --
            Phillip

            Comment


            • #7
              Originally posted by pmiguel View Post
              Are you getting the cluster density from SAV? If not, where?

              --
              Phillip
              I am asking because normally if I saw odd results from a run -- where there were no PF% calculated at all -- I would check the images using SAV. If there were no images, but the run finished on the instrument, then I would check the thumbnail images on the MiSeq itself.

              15pM seems like a crazy high clustering density to me. Since you included 5% phiX, it makes me think your libraries are low diversity? No way you could cluster a low diversity library at 15pM on a v2 cassette and get PF clusters. At least not in my experience. We usually cluster at 5pM (where library concentration is determined by KAPA qPCR) on v2 cassettes.

              So your story makes perfect sense if you clustered at 15pM and got zero % PF clusters. What seems weird is that you got no % PF reading at all. You should get something there, even if that something is "zero".

              --
              Phillip

              Comment

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