# Library quantification and manual normalization of Nextera XT libraries

After the Nextera XT PCR cleanup step, the library is double stranded DNA and can be \*\*\*\*quantified using a fluorometric method, such as Qubit or PicoGreen.

Bioanalyzer traces or qPCR are **not an acceptable** method for quantifying Nextera libraries.\* Although a Bioanalyzer trace is a good method for assessing final library size, it is not accurate for quantification due to a broad sample size distribution.

* qPCR is appropriate only for libraries with a narrow size range. qPCR uses single-sized standards which are not representative of Nextera XT libraries, and a qPCR standard is not available for the large size distribution of Nextera XT libraries.

Quantify samples with Qubit or PicoGreen, and omit the bead-based normalization steps of the protocol if you are planning to manually normalize the libraries.

For manual normalization:

1. Calculate the molarity of each library using the Qubit quantification values and the average fragment length from the Bioanalyzer.
2. Prepare dilutions of each library to the same concentration (C1V1=C2V2)
3. Pool equal volumes of each normalized library (5 ul of each, for example)

**Note:** See the [Best practices for manually normalizing library concentrations](https://support.illumina.com/bulletins/2017/03/best-practices-for-manually-normalizing-library-concentrations.html) document for more information.

Denature and dilute the samples before loading onto the flow cell. At least 2-4 nM of final library is required to denature and dilute libraries in preparation for sequencing on most sequencing instruments.

**Note:** Consult the denature and dilution protocol for the instrument that will be used for the most appropriate recommendations.

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| *For any feedback or questions regarding this article (Illumina Knowledge Article #1145), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000001145%20-%20Library%20Preparation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |


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