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  • NEXTFLEX Cell Free DNA-Seq Library Prep Kit 2.0

NEXTFLEX Cell Free DNA-Seq Library Prep Kit 2.0

NEXTFLEX NGS Library Prep
NEXTFLEX Cell Free DNA-Seq Library Prep Kit 2.0
NEXTFLEX NGS Library Prep
NEXTFLEX NGS Library Prep
NEXTFLEX NGS Library Prep

The NEXTFLEX™ Cell Free DNA-Seq Library Prep Kit 2.0 is built for liquid-biopsy and cell-free fetal DNA testing workflows, transforming just 10 ng of plasma, serum or other biofluids derived cfDNA into sequencing-ready libraries in as little as 2 hours while preserving the native cfDNA fragment profile.

Whether your study targets circulating tumor DNA (ctDNA) or cell-free fetal DNA (cffDNA), the kit’s single-tube end-repair/A-tailing + ligation chemistry minimizes GC bias and yields high-complexity libraries for confident detection of low-frequency variants across GC-rich, AT-rich, and other hard-to-sequence regions.

Libraries are fully compatible with Illumina® (NovaSeq™, NextSeq™, MiSeq™) and Element Biosciences® AVITI™ platforms, and the workflow is automation-ready, with automation compatible protocols available for Revvity liquid-handling workstations.

For large cohort studies, pair the kit with up to 1,536 NEXTFLEX Unique Dual Index (UDI) Barcodes to multiplex thousands of samples per flow cell with minimal index crosstalk. When single-molecule error correction is required, e.g., MRD or ultra-rare variant applications, choose the 96-plex NEXTFLEX UDI-UMI adapters, which embed a 9-bp unique molecular identifier to suppress PCR/sequencing errors and eliminate index hopping.

View product information ビュー related reagents
Feature Specification
Automation Compatible Yes
Product Group DNA-seq

The NEXTFLEX™ Cell Free DNA-Seq Library Prep Kit 2.0 is built for liquid-biopsy and cell-free fetal DNA testing workflows, transforming just 10 ng of plasma, serum or other biofluids derived cfDNA into sequencing-ready libraries in as little as 2 hours while preserving the native cfDNA fragment profile.

Whether your study targets circulating tumor DNA (ctDNA) or cell-free fetal DNA (cffDNA), the kit’s single-tube end-repair/A-tailing + ligation chemistry minimizes GC bias and yields high-complexity libraries for confident detection of low-frequency variants across GC-rich, AT-rich, and other hard-to-sequence regions.

Libraries are fully compatible with Illumina® (NovaSeq™, NextSeq™, MiSeq™) and Element Biosciences® AVITI™ platforms, and the workflow is automation-ready, with automation compatible protocols available for Revvity liquid-handling workstations.

For large cohort studies, pair the kit with up to 1,536 NEXTFLEX Unique Dual Index (UDI) Barcodes to multiplex thousands of samples per flow cell with minimal index crosstalk. When single-molecule error correction is required, e.g., MRD or ultra-rare variant applications, choose the 96-plex NEXTFLEX UDI-UMI adapters, which embed a 9-bp unique molecular identifier to suppress PCR/sequencing errors and eliminate index hopping.

View product information ビュー related reagents
Product variants
Unit Size: 8 rxns
Part #:
NOVA-5150-01
Unit Size: 48 rxns
Part #:
NOVA-5150-02
For research use only. Not for use in diagnostic procedures.
Request more information
NEXTFLEX NGS Library Prep
NEXTFLEX Cell Free DNA-Seq Library Prep Kit 2.0
NEXTFLEX NGS Library Prep
NEXTFLEX NGS Library Prep
NEXTFLEX Cell Free DNA-Seq Library Prep Kit 2.0
NEXTFLEX NGS Library Prep
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Product information

  • Overview
  • Additional product information
  • Specifications
  • Citations

Overview

By simplifying multi-step cfDNA library prep into a robust, streamlined and automation-friendly workflow, the NEXTFLEX Cell Free DNA-Seq Kit 2.0 removes the usual throughput, and indexing bottlenecks and reduces the bias that limit liquid-biopsy, prenatal, and transplant-monitoring studies. The benefits below highlight how the NEXTFLEX Cell Free DNA-Seq Library Prep Kit 2. conserves precious plasma or serum input, scales from pilot projects to population studies, and plugs seamlessly into Illumina® and Element® sequencing pipelines.

  • Low input: robust performance from 10 ng cfDNA, conserving precious liquid-biopsy samples
  • Rapid turnaround: two-hour total workflow with minimal hands-on time accelerates time-to-result
  • High library complexity: optimized ligation chemistry boosts unique read counts and reduces duplicates
  • Minimal bias: uniform genome coverage improves sensitivity for SNVs, indels, CNVs, and aneuploidies
  • Flexible multiplexing: 1,536 UDIs or 96 UDI-UMIs combat index hopping and enable error correction
  • Automation-ready: validated protocols for Revvity liquid-handlers support scalable clinical-research pipelines
  • Platform versatility: proven on Illumina® and Element Biosciences® sequencers for broad lab adoption

Additional product information

Low-Input cfDNA Performance

Generate deep-coverage libraries from just 10 ng of plasma- or serum-derived cfDNA, the amount typically recovered from under 3 mL samples. Our chemistry preserves the native cfDNA fragment profile, which presents 5′-phosphate and 3′-hydroxyl ends typical of nuclease cleavage. That means you can profile early-stage cancers or run cell-free fetal DNA tests on these small input samples without resorting to pre-amplification or whole-genome amplification that can skew variant calls.

Four electropherogram traces illustrating consistent cfDNA library size profiles generated from 10 ng input with the NEXTFLEX Cell Free DNA-Seq Kit 2.0; main peak  around 320 base-pairs corresponding to mono-nucleosomal cfDNA with no adapter-dimer


Figure 1: Electropherograms of cfDNA libraries prepared from 10 ng plasma cfDNA with the NEXTFLEX Cell Free DNA-Seq Kit 2.0 show a clean ~320 bp peak, negligible adapter-dimer, and highly consistent low-input performance.

Accelerated cfDNA Workflow Efficiency

A consolidated, single-tube end-repair/A-tailing step merges multiple enzymatic reactions, while magnetic-bead clean-ups are tuned for fast binding and elution. The result is a roughly 2-hour start-to-finish protocol with minimal hands-on pipetting, short enough to move from sample intake to sequencer load in the same shift. Less bench time frees staff for higher value tasks, reduces pipetting error, and helps labs hit aggressive turnaround targets.

High-Complexity Libraries with Uniform Genome Coverage

Optimized ligation chemistry and precisely calibrated adapter stoichiometry maximize single-insert tagging efficiency, minimizing adapter-dimer formation and PCR duplicates while enriching the pool of unique cfDNA molecules that reach the flow cell. At the same time, a balanced buffer matrix, tuned for salt, PEG, and enzyme concentrations, suppresses sequence-dependent bias, delivering uniform coverage across GC-rich, AT-rich, and repetitive regions from telomere to telomere. Together, the resulting high library complexity and even genome representation drive deeper usable read depth and sharper quantitative resolution, enabling confident detection of low-frequency SNVs, indels, CNVs, and aneuploidies, essential for minimal-residual-disease monitoring, clonal evolution analysis, and other applications that demand analytical sensitivity.

Scalable Multiplexing & Error Correction

Select from 1,536 NEXTFLEX Unique Dual Index (UDI) pairs to pool thousands of samples per flow cell with negligible index crosstalk or deploy the 96-plex NEXTFLEX® UDI-UMI set when single-molecule error correction is needed for ultra-rare variant or MRD applications. Both index formats slot seamlessly into Illumina® and Element Biosciences® workflows, so you can dial in either throughput or precision without altering the core protocol.

Specifications

Automation Compatible
Yes
Product Group
DNA-seq
Shipping Conditions
Dual Temperature
Unit Size
8 rxns

Citations

  • Raman L et al. (2022). Shallow-depth sequencing of cell-free DNA for Hodgkin and diffuse large B-cell lymphoma (differential) diagnosis: a standardized approach with underappreciated potential. Haematologica 107(1):211–220. DOI: 10.3324/haematol.2020.268813
  • Zviran et al. (2020) Genome-wide cell-free DNA mutational integration enables ultra-sensitive cancer monitoring. Nat Med. 26:1114–1124.
  • Brenner et al. (2025) Improved pathogen identification in sepsis or septic shock by clinical metagenomic sequencing. medRxiv preprint 2025.04.04.25324949.
  • Müller J, Hartwig C, Sonntag M et al. (2024). A novel approach for in vivo DNA footprinting using short double-stranded cell-free DNA from plasma. Genome Research 34(8): 1185–1195. DOI: 10.1101/gr.279326.124
  • Mauger F, Horgues C, Pierre-Jean M et al. (2020). Comparison of commercially available whole-genome sequencing kits for variant detection in circulating cell-free DNA. Scientific Reports 10: 6190. DOI: 10.1038/s41598-020-63102-8

FAQs

  • What is the minimum cfDNA input I can use?

    The kit is validated down to 10 ng total cfDNA, allowing reliable library prep from approximately 3 mL of plasma or serum. Basal concentrations of plasma cfDNA in healthy adults generally lie between 1 - 10 ng/mL, with a median around 4 ng/mL.

  • Which sample types work best?

    The chemistry is optimized for plasma- and serum-derived cfDNA and is routinely used for circulating tumor DNA (ctDNA) and cell-free fetal DNA (cffDNA) applications. It also performs well for transplant-monitoring cfDNA; run a small pilot if you’re working with other biofluids such as urine or CSF.

  • How long does the workflow take, and how much hands-on time will I need?

    Library prep finishes in about 2 hours, with less than 30 minutes of active pipetting thanks to a single-tube end-repair/A-tail/ligation step and streamlined bead clean-ups.

  • Can I automate the protocol?

    Yes. Pre-validated scripts for Revvity Sciclone™ G3 NGSx, NGSx iQ™, and Zephyr™ G3 liquid handlers turn the entire protocol, bead washes included, into a walk-away workflow for 96- or 384-sample batches.

  • Which sequencers are supported?

    Libraries load directly onto Illumina® NovaSeq™, NextSeq™, MiSeq™, and Element Biosciences® AVITI™ instruments with no protocol changes.

  • What library size profile should I expect?

    Electropherogram traces show a dominant peak at ~320 bp corresponding to mononucleosomal fraction (cfDNA insert plus adapters), negligible adapter-dimer below 150 bp, and minimal presence of fragments compatible with dinucleosomes at ~480 bp.

  • How do NEXTFLEX UDI-UMI adapters improve data quality?

    The 96-plex UDI-UMI set adds a 9-bp Unique Molecular Identifier to each fragment while retaining dual indexing. Collapsing reads with the same UMI suppresses PCR/sequencing errors and virtually eliminates index hopping, ideal for ultra-rare variants and minimal-residual-disease (MRD) studies.

  • Are the libraries compatible with hybrid-capture panels or shallow WGS?

    Absolutely. Finished libraries can feed directly into hybrid-capture target-enrichment workflows or shallow whole-genome sequencing for CNV and fragmentation analyses without additional modification steps.

Resources

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Application Note
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Streamlining NGS: A comprehensive guide from sample prep to QC
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This flyer showcases Revvity comprehensive WGS portfolio.

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