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  • pmiguel
    replied
    Originally posted by LFMar View Post
    Hi everybody, I just performed an amplicon run to sequence 16S RNA using fusion primers and the "one way read approach". The amplicons have ~700bp and the bioanalyzer profiles were perfect with no primer-dimer peak.
    ssDNA primer-dimers can anneal to the adapters of your 700 bp amplicons and effectively "hide" from size/molecular weight-based fractionation methods. I was hearing that people were getting better results on PCR products (which is what fusion amplicons are) when they were subjected to 2 cycles of AmPureXP clean-up.

    AmPure is a size-based fractionation method, but perhaps by doing 2 cycles the primer-dimer strands exchange off the full length amplicons enough to get a good result.

    Or, you could go old-school and run a denaturing acrylamide gel. That way all the strands run separately and the primer-dimers can't hide.

    --
    Phillip

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  • RCJK
    replied
    Try using the emPCR conditions for long amplicons and see if that helps.

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  • LFMar
    replied
    Originally posted by RCJK View Post
    LFMar, what emPCR conditions did you use?
    I used standard emPCR conditions

    Leave a comment:


  • RCJK
    replied
    LFMar, what emPCR conditions did you use?

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  • LFMar
    replied
    Hi everybody, I just performed an amplicon run to sequence 16S RNA using fusion primers and the "one way read approach". The amplicons have ~700bp and the bioanalyzer profiles were perfect with no primer-dimer peak. Our machine was upgraded to GS FLX+ but we used XLR70 reagents. Guess what? I obtained a high % of short read/short primer, reads with low quality. The processing was made using amplicon and shotgun pipelines giving the same poor result. Amplicon sequencing using 454 shouldn't be allowed. I would appreciate any advise from you. Thanks in advance.

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  • pmiguel
    replied
    I am interested to know if there is some issue with your instrument running Titanium since you got the upgrade or, as I suspect, the runs are fine but the way your IT guys tuned your amplicon processing pipeline is not compatible with Titanium-run-on-a-FLX+ data.

    --
    Phillip

    Leave a comment:


  • pmiguel
    replied
    Hi Indiana,
    When was your instrument upgraded to XL+?
    Any issues with your shotgun runs?

    What parameters are you using for signal processing? (You would probably have to get that info from your IT guys.)

    --
    Phillip

    Leave a comment:


  • 454 amplicon recurring problem - wide, large untrimmed distribution

    Hello,
    We have done several amplicon sequencing projects lately that have yielded poor results. Link below is to a summary of thoses runs including Bioanalyzer traces, untrimmed and trimmed distributions for several of them (pword is "amplicon"):



    Good runs were on:

    April 22
    June 7
    July 31
    Oct 19 (Region 2)

    Then the weird distributions started (wide and too-high untrimmed, dismal trimmed):

    Nov 3
    Nov 7
    Nov 18 (partial)
    Nov 26 (partial)

    We are upgraded to GSFLX-XL+, but are still using regular Titanium kits.

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    Multiomics Techniques Advancing Disease Research
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    New and advanced multiomics tools and technologies have opened new avenues of research and markedly enhanced various disciplines such as disease research and precision medicine1. The practice of merging diverse data from various ‘omes increasingly provides a more holistic understanding of biological systems. As Maddison Masaeli, Co-Founder and CEO at Deepcell, aptly noted, “You can't explain biology in its complex form with one modality.”

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