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  • NEBNext® Ultra™ DNA Library Prep Kit for Illumina®


    The NEBNext® Ultra™ DNA Library Prep Kit for Illumina contains enzymes and buffers that are ideal to convert a small amount of DNA input into indexed libraries for next-generation sequencing on the Illumina platform (Illumina, Inc). The workflow of NEBNext Ultra DNA Library Prep Kit for Illumina is very user-friendly and fast with minimal hands-on time. Each of these components must pass rigorous quality control standards and are lot controlled, both individually and as a set of reagents.

    Ultra DNA Library Preparation Workflow for Illumina

    NEBNext Ultra™ DNA provides high library yields even with low inputs and difficult samples
    All libraries were prepared with 5 ng of input DNA and run on an Agilent Bioanalyzer®.
    A. E. coli genomic DNA libraries. 1: Ladder; 2: Library prepared using NEBNext DNA Library Prep Master Mix Set (E6040); 3: Library prepared using NEBNext Ultra DNA Library Prep Kit (E7370). With 5 ng inputs, library yields are significantly higher using the NEBNext Ultra™ DNA Kit.
    B. FFPE sample. Human lung tumor genomic DNA from a 66-year old male, extracted from a 10-year old FFPE block (Biochain, Inc.). 1: Library prepared using NEBNext Ultra DNA Library Prep Kit (E7370). 2: Input DNA (highly degraded FFPE genomic DNA).
    Read depth correlation shows consistent high coverage for 5 ng - 1 µg input amounts
    Libraries were prepared with various amounts of human IMR90 gDNA (5, 50, and 1000 ng) and PCR amplified. A PCR-free library was also prepared with 1000 ng human IMR90 gDNA. Sequence coverage of 10 kb windows of the human genome was analyzed on Galaxy, and Pearson correlation coefficient (R2 values) were calculated using Microsoft® Excel®. All libraries are highly correlated with each other, independent of DNA input as well as PCR amplification. High coverage was achieved for all DNA input amounts.

    NEBNext Ultra DNA Kits display strong performance with input amounts as low as 5 ng
    Input DNA: IMR90 genomic DNA (human fibroblast).

    PF Mismatch Rate: The rate of bases mismatching the reference for all bases aligned to the reference sequence.

    Percent Duplication: The percentage of mapped sequence that is marked as duplicate.

    Percent Chimeras: The percentage of reads that map outside of a maximum insert size or that have the two ends mapping to different chromosomes.

    This data illustrates the strong performance of the NEBNext Ultra DNA Library Prep Kit for Illumina, even with 5 ng of human genomic DNA input. Mismatch rate, % duplication and % chimeras are all low, and similar, regardless of input amount.

    Functional Validation
    Each set of reagents is functionally validated together through construction and sequencing of a DNA library on the Illumina sequencing platform.

    Lot Control

    The lots provided are managed separately and qualified by additional functional validation. Individual reagents undergo standard enzyme activity and quality control assays, and also meet stringent criteria in the additional quality controls listed on each individual component page

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEBNext End Prep Enzyme Mix-20
    NEBNext End Repair Reaction Buffer-2010X
    Blunt/TA Ligase Master Mix-20
    NEBNext Ligation Enhancer-20
    NEBNext High-Fidelity 2X PCR Master Mix-202X
    NEBNext® Q5® Hot Start HiFi PCR Master Mix-20

    Advantages and Features


    • Input amounts as low as 5ng
    • Fast workflow (<3 hours), with minimal hands-on time
    • Robust, reliable performance
    • Ultra high fidelity amplification with minimized GC bias

    Properties and Usage

    Materials Required but not Supplied

    • 80% Ethanol (freshly prepared)
    • Nuclease-free Water
    • 0.1X TE, pH 8.0
    • 10 mM Tris-HCl, pH 7.5–8.0
    • 10 mM NaCl (optional)
    • DNA LoBind Tubes (Eppendorf #022431021)
    • AMPure® XP Beads (Beckman Coulter, Inc. #A63881)
    • NEBNext Singleplex or Multiplex Oligos for Illumina (NEB #E7350, #E7335, #E7500 or #E7600)
    • Magnetic rack/stand
    • PCR Machine

    Storage Temperature



    1. Can AMPure XP beads used for clean-up steps be left in the PCR reactions?
    2. For what type of sample prep/library construction can I use the NEBNext Ultra DNA Library Prep kit for Illumina for?
    3. What type and how much starting material do I need to use when preparing libraries using the NEBNext Ultra DNA Library Prep Kit for Illumina?
    4. Is size selection required and what is the recommended method?
    5. Can I use the NEBNext Ultra DNA Library Prep Kit for Illumina reagents for preparing libraries for sequencing on the Roche 454 GS FLX & SOLiD 5500?
    6. Can I use the NEBNext Ultra DNA Library Prep Kit for Illumina reagents for preparing libraries for sequencing on the ABI SOLiD 4 instrument or on the Ion Torrent PGM?
    7. Can NEB provide any of the reagents for the cluster generation step or the actual sequencing run?
    8. Are these reagents available in a customized format? Larger format or 96 well format?


    1. Please see manual (NEB #E7370) for protocols


    The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name these document files: manual[Catalog Number].

    Application Notes

    Safety Data Sheet

    The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.