NEXTFLEX Rapid DNA-seq kit 2.0

RevvitySKU: REV-NOVA-5188-01

Size: 8rxn


NEXTFLEX® Rapid DNA-Seq Kit 2.0 for Illumina® Platforms

  • Complete library prep solution, including size selection beads
  • Up to 1,536 UDI barcodes are available seperately
  • Convenient and cost-effective package in a 8, 48, or 96 reaction format
  • Simplified workflow
  • Automation compatible with liquid handlers, including the  Sciclone® G3 NGS/NGSx workstations, Zephyr® G3 NGS workstation, and BioQule NGS System
  • Robust genome coverage
  • Reliable performance with GC-bias and sequencing bias mitigation
  • Low input requirement for genomic DNA down to 1 ng
  • For FFPE samples, use the NEXTFLEX® Rapid XP DNA-Seq Kit
  • Functionally tested with Illumina® sequencing platform


  • NEXTFLEX® End Repair & Adenylation Buffer Mix 2.0
  • NEXTFLEX® End Repair & Adenylation Enzyme Mix 2.0
  • NEXTFLEX® Ligase Buffer Mix 2.0
  • NEXTFLEX® Ligase Enzyme 2.0
  • NEXTFLEX® PCR Master Mix 2.0
  • NEXTFLEX® Primer Mix 2.0
  • NEXTFLEX® Cleanup Beads 2.0
  • Nuclease-free Water
  • Resuspension Buffer


  • 1 ng – 1 µg of fragmented genomic DNA in up to 32 µL nuclease-free water.
  • NEXTflex® barcodes
  • Ethanol 100% (room temperature)
  • Ethanol 80% (room temperature)
  • Covaris System (S2, E210) or other method for DNA fragmentation
  • 96 well PCR Plate Non-skirted (Phenix Research, Cat # MPS-499) or similar
  • 96 well Library Storage and Pooling Plate (Thermo Fisher Scientific, Cat # AB-0765) or similar
  • Adhesive PCR Plate Seal (Bio-Rad, Cat # MSB1001)
  • Magnetic Stand -96 (Thermo Fisher Scientific, Cat # AM10027) or similar
  • Heat block
  • Thermocycler
  • 2, 10, 20, 200 and 1000 µL pipettes / multichannel pipettes
  • Nuclease-free barrier pipette tips
  • Microcentrifuge
  • 1.5 mL nuclease-free microcentrifuge tubes
  • Vortex

The NEXTFLEX®  rapid DNA-seq 2.0 kits contain library prep reagents, including cleanup beads, to prepare 8, 48, or 96 genomic DNA samples for Illumina® sequencing. The shelf life of all reagents is 6 months when stored properly at -20°C. This kit is shipped on dry ice.


Rapid DNA Library Prep for Sequencing on Illumina® Platforms

The NEXTFLEX® rapid DNA-seq 2.0 kit is designed to prepare multiplexed libraries for single or paired-end sequencing using Illumina® platforms. The enhanced NEXTFLEX® kit simplifies workflow by using master mixed reagents and magnetic bead based cleanup, reducing pipetting and eliminating time-consuming steps in library preparation. The NEXTFLEX® rapid DNA-seq kit 2.0 produces library preps with a larger number of unique sequencing reads, allowing for robust genome coverage in challenging GC-rich sequences that can introduce bias.

Figure 1: Libraries prepared with NEXTFLEX® rapid DNA-Seq kit 2.0 show higher yield than libraries prepared with Competitor K’s kit using the same number of PCR cycles from a 250 ng and 1 ng input (A) and robust conversion rate (B).

lexible Multiplexing Options

The NEXTFLEX® adapters are long, annealed adapters containing indexed sequences, which offer an improved multiplexing workflow and flexible experimental setups catered to individual needs. Our barcode portfolio encompass options for single or paired-end multiplex sequencing. The NEXTFLEX® Rapid DNA-seq kit 2.0 reagents are compatible with the NEXTFLEX® Unique Dual Index Barcodes  for up to 1,536 sample multiplexing.

For more information about barcode options compatible with the NEXTFLEX® Rapid DNA-seq kit 2.0, please contact

Automation Compatibility

The NEXTFLEX® Rapid DNA-seq Kit 2.0 is compatible with multiple liquid handlers, including the Sciclone® G3 NGS and NGSx workstations. For low throughput automation, the NEXTFLEX® Rapid DNA-seq Kit 2.0 kit is also now automated on the BioQule™ NGS System which simplifies both library prep AND quantitation. This low-cost, benchtop instrument delivers libraries ready to load into your sequencer with only 15 minutes of hands-on time. For larger volume requirements, customization and bulk packaging is available. For increased flexibility, individual reagents (non-master mixed) are also available. Please contact for further information.

Additional Data between NEXTFLEX® Rapid DNA-Seq 2.0 and Competitor K’s Kit Libraries

Figure 2: Libraries prepared with NEXTFLEX® Rapid DNA-seq 2.0 shows less GC bias than libraries prepared with Competitor K’s kit.

Figure 3: Libraries prepared with NEXTFLEX® Rapid DNA-seq 2.0 shows higher genome coverage than libraries prepared with Competitor K’s kit.

Tech Tips – When to Size Select

Size selection is a critical issue in NGS library preparation. Our blog post below, addresses a number of factors to consider before determining a size selection strategy.

Fragmentation of nucleic acids prior to library construction is required by the majority of next-generation sequencing platforms. Available methods for fragmenting high quality genomic DNA vary in their ability to focus sheared nucleic acids to a tight average fragment size. Broadly distributed shearing profiles are obtained when using probe-based shearing instruments such as QSonica’s sonicator, whereas ultrasonication instruments, such as Diagenode’s Biorupter®ultrasonicators or any instrument from the  product line offered by Covaris, result in much more controlled, tighter shearing profiles. In recent years, enzymatic fragmentation modules have become coupled with downstream library prep to offer a more convenient, automation-friendly offering for labs needing high-throughput solutions. Highly variable shearing profiles of starting material and limited sequencing read lengths leave the researcher with an important question of whether or not to size select NGS libraries.

Read Length Considerations

Read lengths play an important role in determining if size selecting NGS libraries is necessary. If starting with a broad shear profile (100 – 1,500 bp) and performing 2×150 reads, it would be advisable to size select 300 – 400 bp or 350 – 500 bp, post-ligation. This strategy would ensure maximum coverage of most inserts. If size selection is not employed in this scenario, many higher molecular weight molecules will not be sequenced deeply, resulting in non-uniform genome coverage.

Starting Material: Low Quality DNA

Formalin-fixed, paraffin-embedded (FFPE) nucleic acids can be highly degraded and fragmented, a consequence of the nature of preservation. If starting with sub-nanogram quantities of low quality DNA, size selection would not be advised due to the limited number of amplifiable DNA molecules going into PCR, which could result in greatly reduced library yield.

Starting Material: Sufficient Quantity of High Quality DNA

Size selecting a specific region of a broad range shear is advisable if starting with ≥ 10 ng of DNA. If DNA is not a limiting factor and many barcoded samples are being processed in parallel, size selection is highly recommended. If size selection is not performed in this scenario and barcoded libraries are pooled and loaded onto the flow cell for cluster generation, samples containing lower molecular weight DNA will be preferentially amplified via clustering, increasing the number of reads this sample receives and decreasing the number of reads received by other libraries. The same is true for removal of high molecular weight inserts. This is an internal control to ensure each library gets similar reads or coverage.

Size Selecting Peak of Shear

If starting with a Gaussian shear profile ranging from 150 – 600 bp, size selection from 300 – 400 bp would be recommended. This method of selection would produce the highest yielding libraries because this area harbors the largest number of viable sequencing molecules, and thus more successful ligation events and higher yield libraries. Conversely, if selecting outside the peak of the shear, library yields will be lower due to a decrease in the number of viable molecules.

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