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  • Bad runs: Changes in intensities or error rates

    Has anyone seen a drop in signal intensities recently? This seems to have led to lower quality sequence with higher error rates on recent flow cells for us. This has been on four flow cells created on twodifferent cluster stations and run on three different GAs so we are pretty happy to rule out any hardware. This leaves user error (I am happy with my team), chemistry or the flow cells themselves.

    James.

    BTW: does everyone else name their machines? We now have R2D2, C3PO, Yoda, General Cluster and Little Bighorn, the chaps in the lab are going mad running so much sequencing!

  • #2
    James,

    I always thought the GAs looked like Hewey, Dewey and Louie (film :Silent Running 1971 - not the ducks) ?

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    • #3
      Paired end or single end runs? If paired end, we have seen a drop recently and the peltier cooler being out of calibration is the suspect. Haven't seen a change in the single end runs lately but have noticed the PhiX library from Illumina is not performing as well as past aliquots have.
      HudsonAlpha Institute for Biotechnology
      http://www.hudsonalpha.org/gsl

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      • #4
        James, today we are setting up our second paired-end run on the same gDNA samples, both runs done in May 2009. Both runs had low starting intensities (200s for C, 400s for A and 800-900 for T and G. Our usual C and A intensities are above 600. Two different users prepared the flowcells and everything has been double-checked (reagent volumes in the cluster station, GAII, PE module, etc) and no problems have been found.

        In the first run, the final intensities of the first read were very low (in the 100s). The second read started with such low intensities that we just considered it lost. I will see next week about the current run, I am hoping for the best, preparing for the worst.

        Could this be a global issue?

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        • #5
          Just some more observations:

          We recently had two runs in a row, on one of our two GAIIs that had very low intensity (first base average ~85). One was a PE flowcell (indexing) and the other a single-read run. A subsequent run on this same instrument has been fine, however.

          ......we may start calling one of ours "Monica" (sorry, inside joke for any long ago members of the ABRF listserver)

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          • #6
            Yes, I have also been seeing low intensities. On the last two runs the 1st cycle intensities were in the 250s and 100s for g,t and a,c. Which resulted in, very few clusters passing filtering.
            Last edited by buckles; 04-22-2010, 12:15 PM.

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            • #7
              When we do have a flow cell work, the intensities have been low. I am nearly convinced that the problem is due to a different manufacturer of flow cells. A member of our lab looked at the old GAI, the 30-series and the new 42-series under a microscope and each looks significantly different from the other. The new 42-series has beveled edges which make it an extra pain to line up against the pins in the GAII. I am also thinking that the profile changed as well so that the manifolds don't fit as well in the cluster station.

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