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  • Sequence of PhiX MiSeq primers

    Newbie trying to track down source of failed (~10% of clusters pass filter) MiSeq amplicon sequencing run. I want to look at possible interactions between our expected sequence/custom primers with the primers used to sequence PhiX (spiked at 10% in our run). Can someone point me to the sequence of those primers? Are they based on the adapters or??
    Thanks

  • #2
    How did you set up your run? Was it for custom primers or the primers included in the cartridge? Where did you put your custom primers in the cartridge?

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    • #3
      It was run through a central lab on campus, not by me. We have a low diversity amplicon so it was spiked with 10% PhiX. My understanding (which may well be wrong) is that our custom primers were added to the phiX primers at some ratio(??) and they were multiplexed in the run. I was curious about the sequence of the primers used to prime the phiX library and the possibility of unwanted interaction with our custom primers. Thanks for any suggestions.

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      • #4
        What was your cluster density for this run, and what percentage of your run aligned to phiX?

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        • #5
          Cluster density was 641k but only about ~10% passed filter. 55% of runs aligned to PhiX. Q score on second index run was very low and run halted before/during the second sequence run. Low score of second index is puzzling; we quantified library with qubit and then qPCR so we're reasonably sure of the quantity. Our tech did the qPCR and diluted with intent to get 800k clusters based on past experience with low diversity libraries, so he wasn't too far off. We are basically following 16s protocol (Kozich), just replacing gene-specific ends of primers with our own.

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