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 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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