Nextera XT DNA Library Prep Kit FAQs

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  • Library Evaluation


  • The Nextera XT protocol is optimized for 1 ng of input DNA total. Illumina strongly recommends quantifying the starting genomic material. Nextera XT library prep kits use an enzymatic DNA fragmentation step and thus can be more sensitive to DNA input compared to mechanical fragmentation methods. The ultimate success of the assay strongly depends on using an accurately quantified amount of input DNA library. Therefore, the correct quantitation of the DNA library is essential.

    To obtain an accurate quantification of the DNA library, quantify the starting DNA library using a fluorometric-based method specific for duplex DNA, such as the Qubit dsDNA BR Assay system. Use 2 μl of each DNA sample with 198 μl of the Qubit working solution for sample quantification. Avoid methods that measure total nucleic acid content (e.g., nanodrop or other UV absorbance methods) because common contaminants such as ssDNA, RNA, and oligos are not substrates for the Nextera XT assay.

    Although most amplicons of interest are not likely to be high GC-content, coverage of high GC-content amplicons might have more variability compared to other amplicons.

  • Sequencing


  • Yes, the Nextera XT DNA Library Prep Kit is compatible with any Illumina sequencing instrument. When sequencing on a HiSeq X or HiSeq 3000/4000 patterned flow cell, Nextera XT DNA libraries might perform with relatively lower %PF due to their longer fragment size. However, these instruments typically provide more data due to the higher output.

    A TruSeq Dual Index Sequencing Primer Box PE (PE-121-1003) or TruSeq Dual Index Sequencing Primer Box SR (FC-121-1003) is needed to sequence Nextera XT libraries on a HiSeq 2500 using TruSeq v3 chemistry. Use the PE box with paired-end flow cells and the SR box with single-read flow cells.

    Other HiSeq 2500 chemistry and other systems do not require this additional box.

    Most sequencing systems require 2–4 nM denatured library. For instructions, see the denature and dilute instructions for your instrument.

    Illumina does not support, and strongly advises against, running libraries prepared with different library prep kits in the same lane of a flow cell. Running libraries prepared by different library prep kits in different lanes of the same flow cell or spiking in Illumina PhiX control library in the same lane as any user-prepared libraries is supported.

    If different library types are run in different lanes, dual index recipes and the Dual Index Primer Box are required. The indexes for TruSeq HT and Nextera are unique and not shared.

    For paired-end flow cells, dual indexing now requires 23 additional cycles of sequencing - eight cycles for the Index 1 (i7) Read, eight cycles for the Index 2 (i5) Read, plus seven non-imaging, chemistry-only cycles at the beginning of the Index 2 (i5) Read. For single-read flow cells, dual indexing only requires 16 additional cycles of sequencing - eight cycles for the Index 1 (i7) Read and eight cycles for the Index 2 (i5) Read.

  • Analysis


  • The appropriate analysis tool depends on the application. MiSeq Reporter includes various analysis workflows, including amplicon, assembly, metagenomics, and resequencing. BaseSpace Sequence Hub also offers various apps for de novo assembly, metagenomics, resequencing, and so on.