NEXTFLEX® Cell Free DNA-Seq Library Prep Kit 2.0 for Illumina® Sequencing Platforms

  • Designed for low sample input – 10 ng of input cfDNA required
  • Accelerated workflow with minimal hands-on time
  • Flexible adapter barcode options – up to 1,536 unique dual index barcodes are available
  • Compatible with Illumina® sequencing platforms
  • Protocols available for automation on the Sciclone® G3 NGSx, NGSx iQ™ and Zephyr® G3 NGS workstations

  • NOVA-5150-01

    8 RXNS

  • NOVA-5150-02

    48 RXNS
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For research use only. Not for use in diagnostic procedures.

Prepare Libraries in Only 2 Hours

The NEXTFLEX® Cell Free DNA-Seq Kit 2.0 can produce libraries from 10 ng of cell free DNA for circulating tumor DNA (ctDNA) or cell free fetal DNA (cffDNA) analysis, in two hours. This low-input library preparation kit delivers high coverage quality and reduced bias for Illumina® sequencing applications. The NEXTFLEX® Cell Free DNA-Seq Kit 2.0 can be used to prepare single, paired-end and multiplexed DNA libraries for sequencing using Illumina®platforms. The availability of up to 1,536 unique dual index barcodes facilitates high-throughput applications without compromising data integrity.

We recommend using the NextPrep-Mag cfDNA Automated Isolation Kit for isolation of circulating cfDNA from blood plasma prior to library prep with the NEXTFLEX® Cell Free DNA-Seq Kit 2.0.

Reduced Bias and Coverage Gaps

NEXTFLEX® Cell Free DNA-Seq Kit 2.0 exhibits minimal GC bias and produces uniform coverage of difficult to sequence genome regions. This kit also offers robust ligation efficiency, resulting in greater library diversity and a larger number of unique sequencing reads. These optimizations enable investigators to obtain more information from each sequencing run.

Flexible Multiplexing Options

The NEXTFLEX® Cell Free DNA-Seq Library Prep Kit 2.0 for Illumina®  sequencing platforms is designed to be used with the NEXTFLEX® Unique Dual Index barcodes (384 barcodes) or the NEXTFLEX® Unique Dual Index barcodes (1,536 barcodes). Thesed NEXTFLEX® Adapters are long, annealed adapters that offer an improved multiplexing workflow and flexible setup.

Automation Solutions

This kit is automated on the Sciclone® G3 NGSx, NGSx iQ™ and Zephyr® G3 NGS workstations. Contact us at [email protected] for more details.


  • NEXTFLEX® cfDNA Enrichment Beads
  • 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 (50 μM)
  • Nuclease-free Water
  • NEXTFLEX® Resuspension Buffer
  • NEXTFLEX® Cleanup Beads 2.0 1.5 mL


  •  ~1 ng of Cell-Free DNA in up to 32 µL nuclease-free water.
  • NEXTFLEX® DNA Barcodes – 6 / 12 / 24 / 48 (Cat # 514101, 514102, 514103, 514104) or NEXTFLEX-96 DNA Barcodes (Cat # 514106)
  • Ethanol 100% (room temperature)
  • Ethanol 80% (room temperature)
  • 96 well PCR Plate Non-skirted (Phenix Research, Cat # MPS-499) or similar
  • 96 well Library Storage and Pooling Plate (Fisher Scientific, Cat # AB-0765) or similar
  • Adhesive PCR Plate Seal (BioRad, Cat # MSB1001)
  • Magnetic Stand -96 (Ambion, Cat # AM10027) / or / similar
  • Thermocycler
  • 2, 10, 20, 200 and 1000 µL pipettes / multichannel pipettes
  • Nuclease-free barrier pipette tips
  • Vortex

NEXTFLEX Cell Free DNA-Seq Workflow

Cell Free DNA-Se enrichment data

Figure 1. Nucleosome enriched libraries

10 ng input of cfDNA was used for the enrichment (or no enrichment) and prepared using the NEXTFLEX® cell-free DNA-seq kit 2.0 standard protocol with the following enrichments.

  • A) No enrichment
  • B) Mono-nucleosome enrichment
  • C) Mono-, di-, tri-nucleosome enrichment

cell-free DNA-seq kit 2.0 libraries

Figure 2. Automated NEXTFLEX® cell-free DNA-seq kit 2.0 libraries

cfDNA was isolated from 5 mL of plasma using the NextPrep-Mag cfDNA automated isolation kit on the chemagic 360 instrument and cleaned/concentrated to be eluted in 32 µL into the NEXTFLEX® cell-free DNA-seq kit 2.0 using 9 cycles of PCR (without nucleosome enrichment. Libraries were diluted 1:3 and run on the LabChip® GXII Touch HT nucleic acid analyzer.

Papers that Cite the Use of the NEXTFLEX® Cell-Free DNA-Seq Kit:

  • Xia, S. et al. (2015) Genomic variations in plasma cell free DNA differentiate early stage lung cancers from normal controls. Lung Cancer. doi: 10.1016/j.lungcan.2015.07.002.
The NEXTFLEX®  Cell Free DNA-Seq Kit 2.0 contains enough material to prepare 8 or 48 DNA samples for Illumina® compatible sequencing when bundled with the NEXTFLEX® NGS barcodes. The shelf life of all reagents is 6 months when stored properly. The Resuspension Buffer and Nuclease-free Water can be stored at room temperature. The NEXTFLEX® cfDNA Enrichment Beads and Cleanup Beads 2.0 should be stored at 4°C, and all other components should be stored at -20°C.
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