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  • elizondas
    Member
    • Nov 2013
    • 14

    454

    I would like to sequence 16s from more than 400 sample of nasopharyngeal samples by pyrosequencing.

    It seems to be a good method to do that saving time and resources. Does anyone have any experience in this? Someone with a direct experience in issues as sample preparation, quality control etc

    We want to use 27F-534R primers to sequencing V1-V3 region. I had thought to add them adaptor A and B, and then a Broad Institute bardcode. I am not sure if I need one bardcode per sample, and therefore I would needed >400 bardcodes between 7-12 pb. I suposed a linker is neccesary in all design primers.

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  • GATTACAT
    Reply to Nine Things a Sample Prep Scientist Thinks About Before Sequencing
    by GATTACAT
    Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.
    07-01-2026, 11:43 AM
  • SEQadmin2
    Nine Things a Sample Prep Scientist Thinks About Before Sequencing
    by SEQadmin2


    I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.

    Here are nine questions we think about, in roughly the order they matter, before...
    06-18-2026, 07:11 AM

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