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454 Run Failure, emPCR and Libraries okay?



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  • 454 Run Failure, emPCR and Libraries okay?

    Greetings, forum. I am new here and I have searched for this problem in previous threads and have not found it. If this has already been discussed, I apologize for my redundancy and feel free to link me to the thread.

    If not, I'm wondering if anyone can help me understand what may have gone wrong with my sequencing run.

    I am doing multiplex sequencing of 23 barcoded 1000bp PCR amplicons of the V1-V5 region of the bacterial 16S rRNA gene. We don't do our own sequencing here ... another facility on our campus does it for us so I am unsure of the technical specifics. They provided us with the barcoded primers and we tested them numerous times before generating the libraries. Still in learning mode. This is what they told us about our run:

    "The library was the correct size based on the Bioanalyzer data.
    And, the enrichment was good for the emulsion PCR."

    My libraries had a low A260/A230 ratio, but they told me that since the libraries are diluted 1000x before the emPCR so any inhibitor would have been negligible.

    This is what else they told us:
    "The sequencing did start and you can see the failed sequences in flowgrams in the GS RunBrowser."
    "We ran 2 other samples using libraries we made with 16s barcoded primers and the ePCRs were fine with those."

    Additionally, my adviser asked this question:
    "By indicating that the library was the correct size, doesn't that mean that there is an insert? If that is the case, then it seems that there must be a problem with the primers [eg that the 454 primer site wasn't present?]
    We got these from you so that shouldn't be the problem. We cloned from the PCR and got nice 16S sequence."

    Feel free to ask me any questions you need to clarify more about this problem. Thank you in advance for any help you can offer!

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