Nextera DNA Flex Kit FAQs

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  • General


  • Nextera DNA Flex library prep kit provides the flexibility of use across a wide range of genomes from small genomes (microbial genomes, amplicons) to more complex genomes (eukaryotes and human). 

    Nextera DNA Flex library prep is rooted in an innovative bead-linked transposome (BLT) complex that tagments genomic DNA by fragmenting and adding adaptor tag sequences in a single reaction step. Once saturated with input DNA, the BLT complex fragments a set number of DNA molecules, providing flexibility to use a wide DNA input range, consistently tight fragment size distribution, and normalized libraries. Following the tagmentation step, a limited-cycle PCR step adds Nextera DNA Flex-specific index adapter sequences to both ends of the DNA fragment, enabling single- and dual-indexed sequencing of pooled libraries across all Illumina sequencing platforms. A subsequent Sample Purification Bead (SPB) cleanup step then prepares libraries for use on an Illumina sequencer.

    Although they share sequence compatibility, it is not recommended to use XT indexes with Flex libraries as Nextera DNA Flex has indexes of higher purity that have been carefully optimized for improved performance. 

    No, Nextera DNA Flex reagents are not compatible with Nextera DNA or Nextera XT reagents as the enzymatic chemistry of DNA Flex is very different from that of Nextera DNA and Nextera XT.

    Normalization in Nextera DNA Flex happens during tagmentation, hence there is no need to perform bead-based normalization at the end of library prep. Furthermore, the Nextera XT bead-based normalization process is not compatible for use with Nextera DNA Flex.

    No. Nextera DNA Flex chemistry is incompatible with the enrichment chemistry.

    The kit includes a sufficient volume of Sample Purification Beads reagent to process the intended number of samples for the kit. Do not use beads or columns from any other manufacturer with this protocol.

    Coverage of GC regions can be impacted by the model, settings, and performance of the thermal cycler used. Illumina has validated the Bio-Rad DNA Engine Tetrad 2, the Bio-Rad S1000, the Bio-Rad C1000, and the MJ Research PTC-225 DNA Engine Tetrad thermal cyclers. Other thermal cyclers may differ in their performance which may impact genomic coverage.

  • Library Evaluation


  • When using DNA input of 100–500 ng, the library prep normalization features obviate the need to quantify and normalize individual libraries generated within one experiment.

    However, you might observe slight variations in the final yields between different library preparation events. To achieve optimal cluster density, pool the libraries with equal volume and quantify the pool.

    Because variations between library preparation events and users are possible, individually quantify and normalize the libraries when pooling samples from different experiments.

    Use an Agilent Technologies 2100 Bioanalyzer to check the quality and intended size distribution of a tagmented sample, the pre-enriched library, and the post-enriched library. For examples of bioanalyzer traces and library size distributions, see the library prep reference guide. Variation in the Bioanalyzer profiles is expected because it is dependent on the input DNA type.

    The profile of the amplified library product can look different from the example shown depending on the type and quality of input DNA. However, the use of BLT and a double-sided size selection helps in maintaining the product length.  However, inaccurate pipetting during the double-SPRI will cause fragment size variation.  Some variation is expected in the profiles of different libraries.

    It is not atypical to see a small bump/shoulder/peak at 1000-3000 bp.  The appearance of the peak can vary between DNA samples and does not affect downstream sequencing. We do not know what this peak is and data is unaffected by this peak. 

    600 bp average fragment length on Bioanalyser (150-1500bp size range).

    350 bp median insert size from sequencing.

    Actual yield may vary based on sample quality and sample type, typically for good quality DNA sample the expected library yield is greater than 4 ng/ul and within an experiment the CV of library yield is <20%.

  • Automation


  • Methods are written and tested by our automation partners to support both 24 & 96 sample kit configurations, including a full plate run with up to 96 reactions.  The 96-sample kit is designed to be automation-friendly, including additional volumes necessary for automated processing.

    No, automation is only supported by the 24 plex and 96 plex Index plates provided by the Nextera DNA flex kit.

    Methods designated "Illumina Qualified" are developed, distributed, and supported by our automation partners. Visit High-Throughput Library Prep Automation to learn more about the partnership program and partners.

    In general, Nextera DNA Flex is designed to be compatible with most automated liquid-handling systems.

    All Illumina automation partners are developing methods for at least one existing automation platform. Contact the individual partners for more information. To view a list of partners, see High-Throughput Library Prep Automation.

    All reagents provide sufficient overage volume to support automation requirements in the 96-sample kit configuration.  Additionally, the 96-sample kit configuration should support at least two runs of 48 samples each (2 runs x 48 samples).

    We recommend utilizing Illumina Qualified* methods from our partners, benefits include:

    • Reduced development & implementation costs.
    • Utilize tested & standardized methods.
    • Choose solutions from leading vendors in laboratory automation.

    *Illumina Qualified indicates that libraries prepared with this method have been shown to perform comparably to those prepared manually.