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Looking for a standardized method of library quantification and quality control

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  • Looking for a standardized method of library quantification and quality control

    Hello all,

    I am wondering if anyone out there has discovered/created a standardized method for library quantification and quality control that works for them.

    The issue that I keep running up against is that, depending on the method used for library preparation, the values given by tapestation, qbit, qpcr and fragment analyzer can vary wildly. Sometimes, for well prepared libraries I will get good consensus between the instruments. But when I don't, it turns quantification into a completely heuristic process.

    I have observed the following trends:
    10x libraries are better quantified on tapestation than qubit,
    Amplicons and Nextera libraries don't seem to work well on tapestation OR qubit,

    fragment analyzer and qpcr are both time-expensive, and the tapestation is just expensive.

    With all that said, has anyone out there been able to completely standardize the process of QC and library quant for all library types? Thank you in advance.

  • #2
    We have NextSeq500/550, I have tried many lib quantification methods (Qubit, NanoDrop, tapestation, Quantfluor and qPCR), the only one works to me is qPCR with kit from KAPA. I have run many libraries constructed with different commercial kits, lab-protocols as well, all work with good cluster density and quality.

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    • #3
      Something (Tape/BioA/FragA) for size estimation followed by KAPA qPCR for absolute quantification. That's the gold standard in my opinion. If it hasn't been qPCR'd we don't run it!

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