Library quantification and manual normalization of Nextera XT libraries

The Nextera XT protocol provides two methods for normalizing final libraries, bead-based normalization and manual normalization. This article focuses on manual normalization, and best practices for quantification.

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 acceptable methods for quantifying Nextera libraries.

• Although a Bioanalyzer trace is recommended for assessing final library size, it is not accurate for quantification due to the broad library size distribution of Nextera XT libraries.

• 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 broad size distribution of Nextera XT libraries.

Quantify samples with Qubit or PicoGreen, and omit the bead-based normalization steps of the protocol if 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 nanomolar concentration (C1V1 = C2V2)

3. Pool equal volumes of each normalized library (5 µl of each, for example)

See Best practices for manually normalizing library concentrations for more information.

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

For any feedback or questions regarding this article (Illumina Knowledge Article #1145), contact Illumina Technical Support [e-mail].