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Library Quatitation: Science or Voodoo

February 8, 2012

If you call Illumina about problems with library quantitation, they won’t give you a straight answer. They aren’t hiding anything; they just don’t have a good answer. Libarary quantitation is a little bit science and a little bit voodoo. The three main methods used to quantitate libraries are nanoDrop, Qubit and qPCR. NanoDrop is said to be sensitive to contaminants and thus not recommended. The QubitHS DNA assay kit from LifeTech is highly specific to dsDNA, which is actually a problem because significant portions of your library may be single-stranded, bubble shaped molecules or daisy chains of molecules because of the high complexity of the library insert and the low complexity of the adapter sequences on the end. This is especially a problem if your library is over amplified (see the previous post on how to avoid this). Both of these methods quantitate total DNA and are not specific to your adapter ligated DNA. That leaves qPCR, which also has its disadvantages but in my opinion they are less than the other two methods. The main problem with qPCR is that different libraries may amplify with different efficiencies and thus cause you to under or over-estimate your library concentration. Ideally, your standards would be identical to your library but that is obviously not possible. The easiest way to qPCR quantitate your libraries is order the library quantification kit from KAPA Biosystems. The kit is basically serial dilutions of a library and some primers. So this is obviously something that you can make yourself if you have a library you are confident of the concentration (see my library quantitation protocol), but you probably would not be reading this post if you did. So in conclusion this is my advice:

1) Quantitate your library with qPCR (this is the science half).
2) Since it is better to get fewer cluster of high quality than a lot of cluster with low quality, use a little less than the amount Illumina recommends
3) After you run your samples make a note of whether your cluster density was too low or too high (this is the voodoo half).
a) If your cluster density was too high, next time around use less library.
b) If your cluster density was too low, next time around use more library.

Final note: It is very important at the final step of your library preparation, after PCR amplification, that you purify your library with AMPure beads to remove the unused PCR primer. Unused PCR primer interferes with the binding of your library to the flow cell (A large genome center’s improvements to the Illumina sequencing system).

Final final note: In theory you can use the bioanalyzer for library quantitation. Don’t do this. Use it to check the size of your libraries and that you have no adapter dimers, but don’t use it for quantitation.

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2 Comments
  1. Benjamin Rodriguez permalink

    Amen, brother. Testify. Do you have any descriptive statistics of cluster numbers (pass filter) relating different library fragment sizes and GC contents to your Kapa library standard curve qPCR? I know the Broad is using Kappa in house. But, I don’t know how consistent it can perform across different size / GC compositions. I have had to listen to the voodoo of which you speak for far too long.

    So there is money to be had by scientists in Australia? What’s the income tax rate?

  2. ethanomics permalink

    I just prepare the libraries. I don’t actually do the sequencing which makes the whole thing even more mysterious. I hope the Broad gets a good deal on the Kapa standards because they are a lot of money to spend for someone to do a PCR reaction for you.

    The pay scale is definitely tilted in our favor in Australia as compared to the US. I don’t know much about their tax code. Hopefully, it’s less corrupt and full of loopholes then the US tax code and better enforced then the Greek tax code. What nice is I don’t have to sped $19,000/year for health insurance for my family, which is the average cost in the US. The killer in Australia is the cost of housing. It makes San Francisco look like Detroit. They must be sitting on the mother of all housing bubbles. For now with lots of austerity in Europe and talk of it in the US, it looks like a good option.

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