One-tube enzymatic fragmentation - libraries in as little as 2.5 hours

The NEXTFLEX enzyme cocktail performs fragmentation, end-repair, and A-tailing in a single tube. A complete manual run, from DNA to normalized libraries, takes as little as 2.5 hours total. Fragment size is tunable by adjusting the 35°C incubation. This streamlined chemistry removes the need for sonication equipment, cuts out an extra cleanup step, and reduces thermocycler runs.

Broad input range WGS with PCR-free option

The protocol accepts 100 pg – 1 µg of genomic DNA. For ≥ 250 ng inputs, users may skip PCR (Appendix A workflow), preserving library complexity and minimizing duplicates; low-input samples use 2–15 cycles as recommended. Expected post-PCR yield is 100–500 ng, sufficient for one or more flow-cell lanes.

Highly Efficient Ligation with Minimal Adapter-Dimer Formation

The NEXTFLEX Rapid XP V2 DNA-Seq Kit delivers exceptional ligation efficiency in a single 20°C incubation.

• 30-minute incubation – keeps total library-prep time at 2.5 hours for time-sensitive runs.
• 60-minute incubation – produces optimal ligation efficiency when the highest possible conversion is critical, for example, PCR-free workflows or ultra-low-input samples.

Optimized reagents join adapters without a prior dilution step, converting nearly all fragments across our entire input range. Electropherograms from 500 pg and 1 ng libraries (Figure 1) show no detectable adapter-dimer peak (~150 bp), even at picogram inputs. This combination of high ligation yield and low dimer formation increases usable sequencing reads and ensures reliable, reproducible data.

Figure 1. Electropherogram traces for libraries prepared from 500 pg (A) and 1 ng (B) human gDNA with the NEXTFLEX Rapid XP V2 DNA-Seq Kit. The dominant library peak is flanked by an adapter-dimer region at ~150 bp that is virtually absent, confirming highly efficient ligation and cleanup even at sub-nanogram inputs.

Uniform coverage across the GC spectrum

GC-bias analysis shows flat, normalized depth from ~20% to 60% GC, independent of input mass; only mild drop-off appears above 60%. This consistency yields near-complete genome coverage at standard read depths, boosting SNP/indel sensitivity without expensive over-sequencing.

Figure 2. Normalized coverage across varying %GC content for the 8 replicates analyzed. The minimal GC bias observed indicates a high degree of coverage uniformity in the data generated.

Built-in NEXTFLEX Normalization Beads - save ≈ 3 h per 96-sample batch

Proprietary beads bind a fixed mass of library DNA during the final cleanup, so every sample leaves the protocol at the same molarity. This single step simplifies the workflow, reduces hands-on time, and ensures optimal library-input balance for precise pooling and sequencing. By eliminating post-library qPCR/fluorometric quantitation and manual pooling, labs can save up to three hours on a 96-sample run. The on-bead normalization also cuts sample-to-sample variability, preventing costly resequencing due to over- or under-clustering and eliminates the need for quantitation reagents and instrumentation.

Figure 3. Workflow comparison of traditional post-library normalization versus built-in NEXTFLEX bead normalization. The conventional route requires three downstream QC steps, quantify libraries, fragment sizing, dilute & pooling, adding ~3 hours to turnaround, while NEXTFLEX beads perform normalization during library prep with no extra time.

Indexing options - from duplex-error correction to population-scale multiplexing

NEXTFLEX indexing chemistry is modular, letting you choose the depth of error control and the level of multiplexing your project demands:

Automation-ready workflows

Revvity provides vendor-qualified scripts for Sciclone™ G3 NGSx/iQ, Zephyr™ NGS, and Fontus™ workstations, so users can drop the NEXTFLEX onto pre-mapped deck positions and run a validated protocol - no custom coding or tip-mapping required.

The same normalization chemistry, available as a standalone

Need extra normalization capacity or want the same on-bead normalization used in Rapid XP v2 across other runs or plates? NEXTFLEX NGS Library Normalization Beads provide pool-ready libraries at a consistent molarity right after PCR, which helps standardize loading across large batches and mixed workflows

• Guo, F., Liu, R., Pan, Y., Colasanto, M., Collins, C., & Hegde, M. (2024). Beyond single diagnosis: Exploring multidiagnostic realities in pediatric patients through genome sequencing. Human Mutation, 2024, 9115364. Beyond Single Diagnosis: Exploring Multidiagnostic Realities in Pediatric Patients through Genome Sequencing
• Di Martino, M. L., Jenniches, L., Bhetwal, A., Eriksson, J., Lopes, A. C. C., Ntokaki, A., … Sellin, M. E. (2025). A scalable gut epithelial organoid model reveals the genome-wide colonization landscape of a human-adapted pathogen. Nature Genetics. Advance online publication. A scalable gut epithelial organoid model reveals the genome-wide colonization landscape of a human-adapted pathogen