Balanced by design
Library quantification and normalization remain a persistent bottleneck for NGS labs, introducing variability at a critical point in the workflow. This step is prone to error: double dipping, skipped wells, and inaccurate serial dilutions of high-concentration libraries often lead to uneven representation or “jackpotting” in pooled samples. The result is inefficient sequencing at best, or rework and resequencing at worst, which disrupts timelines, increases costs, and adds operational complexity.
EquiPlex™ is designed to address these challenges directly, leveraging Cas9 in a fundamentally different way than its genome editing roots — not to cut, but as a programmable, stoichiometric binding tool that enables more consistent, reliable normalization to support smooth, scalable sequencing workflows.
Key Features and Benefits
- Cas9-based approach delivers uniform sample coverage across a 5 – 200 nM input range while preserving library integrity
- Fast, automatable workflow processes up to 96 libraries in under one hour with no manual interventions or off-deck handling steps
- Eliminates manual dilution calculations, variable-volume pipetting, and library jackpotting, ensuring every library receives its fair share of sequencing reads
- Supports singleplex¹ and multiplex² workflows with flexible batch sizes from 8 to 96 libraries
- Requires only half of your amplified library, leaving the remaining volume available for future use
1. EquiPlex Normalization Kit (Singleplex)
2. EquiPlex Normalization Kit (Multiplex)
Applications
- RNA sequencing, including 3’ sequencing
- Methylation sequencing
- DNA sequencing, including ChIP-seq and metagenomics
- Targeted sequencing (post-capture)
- Single cell sequencing (non-tagmentation workflows)
- Amplicon sequencing
Designed for
- Amplified libraries built with TruSeq®-style adapters
- Libraries moving directly to sequencing, including post-hybrid capture
Cas9-mediated Library Normalization
Figure 1. Rapid, automatable normalization of NGS libraries. EquiPlex Normalization Kits leverage Cas9 to normalize libraries without degrading or single-stranding them. Both configurations — the Singleplex Kit, which normalizes libraries individually, and the Multiplex Kit, which normalizes libraries in pools — tag a fixed number of molecules with a Cas9-mediated binding reagent and use bead-based capture to select for bound molecules. The entire process takes less than one hour manually, and is fully automatable, with no manual interventions or off-deck steps required.
Mix and Match Pool Sizes to Fit Your Experiment
The EquiPlex Normalization Kit (Multiplex) supports pool sizes from 8 to 96 libraries per batch, with reagents included for up to 96 total libraries per kit. Different pool sizes can be combined within a single run, giving you the flexibility to match any experimental design.
Figure 2. Flexible pooling configurations to fit any experimental design. The EquiPlex Normalization Kit (Multiplex) allows libraries from a single 96-well plate to be grouped into pools of any size from 8 to 96 libraries — shown here as 8-, 16-, 24-, and 48-plex — with no requirement for spatial adjacency on the plate and no restriction to multiples of 8. Any combination of pool sizes can be used from a single kit, up to 96 total libraries.
A Better Way to Normalize
Compared to quantify-and-dilute approaches, EquiPlex is the only normalization solution that combines fast turnaround, full automation, and low readcount variability without manual calculations, specialized equipment, or off-deck handling.
Table 1. Comparison of EquiPlex normalization to quantify-and-dilute methods
| EquiPlex Normalization | Quantify-and-Dilute Methods | ||||
| qPCR | MiSeq/iSeq | Fluorimetric¹ | Electrophoretic² | ||
| Fast turnaround time | +++ | – | – | ++ | – |
| Automatability, including no off-deck handling | +++ | – | – | ++ | – |
| Low readcount variability | +++ | ++ | +++ | + | + |
| No manual calculations, dilutions, or variable volume pipetting | +++ | – | – | – | – |
| No additional or specialized equipment | +++ | – | – – | – | – |
1. Examples: Qubit®, Picogreen®
2. Examples: TapeStation®, Fragment Analyzer®
Key Performance Data
More uniform pools for more efficient sequencing
EquiPlex delivers accurate normalization across a broad input range (5 – 200 nM), enabling uniform read distribution even when pooling a full 96-well plate in a single multiplexed run.
That read uniformity translates directly into sequencing efficiency. More evenly represented libraries mean greater multiplexing confidence, less top-off sequencing and rework, and more output from every flow cell.
Figure 3. Robust normalization across a wide library ... MORE
More uniform pools for more efficient sequencing
Figure 3. Robust normalization across a wide library concentration range. mRNA libraries spanning a 40-fold concentration range (5 – 200 nM, 12 replicates per concentration) were normalized in a single 96-plex batch using the EquiPlex Normalization Kit (Multiplex). Normalized read contributions were approximately 1% per library across all input concentrations, consistent with the expected value for a well-normalized 96-plex pool (100% ÷ 96 = 1.04% per library).
Consistent, reliable normalization for DNA-seq workflows
Uniform library representation means more confident multiplexing, less top-off sequencing, and more output from every flow cell. Across a 500-fold input range into library prep, EquiPlex delivers consistently low read count variability without affecting insert size, GC bias, or data quality, as well as entirely eliminates jackpotting to ensure every sample receives its fair share of reads.
Consistent, reliable normalization for DNA-seq workflows
Seamless integration into RNA-seq workflows
EquiPlex is readily incorporated into RNA-seq workflows, delivering uniform read distributions without compromising data integrity. Gene expression profiles show excellent concordance between EquiPlex and manual normalization, with no measurable effect on percent alignment, gene body coverage, or unique genes detected.
Seamless integration into RNA-seq workflows
BROCHURES
EquiPlex Normalization Kits Brochure
Illumina DNA RNA Blood Cell Line Dried Blood Spot FFPE Frozen Tissue Microbial Plasma/cfDNA Plasmid Saliva/buccal Tumor Tissue Urine DNA NGS Epigenomics RNA NGS EquiPlex NormalizationThe EquiPlex™ Normalization Kits eliminate quantification bottlenecks and pooling variability using a Cas9-based approach that tags a fixed number of library molecules for bead-based capture — no dilution calculations, no variable-volume pipetting, no jackpotting. The fully automatable, sub-one-hour workflow processes up to 96 libraries across a 5–200 nM input range, delivering consistently low read count variability without affecting insert size, GC bias, or downstream data quality.
PROTOCOLS
EquiPlex Normalization Kit (Singleplex) User Guide
Illumina DNA RNA Blood Cell Line Dried Blood Spot FFPE Frozen Tissue Microbial Plasma/cfDNA Plasmid Saliva/buccal Tumor Tissue Urine DNA NGS Epigenomics RNA NGS EquiPlex NormalizationProtocol for the EquiPlex Normalization Kit (Singleplex), which enables the individual normalization and pooling of 96 amplified NGS libraries without prior quantification or dilution. The Cas9-based binding workflow preserves double-stranded library integrity and accepts inputs from 5 to 200 nM, with libraries eluted ready for direct sequencing on Illumina platforms.
EquiPlex Normalization Kit (Multiplex) User Guide
Illumina DNA RNA Blood Cell Line Dried Blood Spot FFPE Frozen Tissue Microbial Plasma/cfDNA Plasmid Saliva/buccal Tumor Tissue Urine DNA NGS Epigenomics RNA NGS EquiPlex NormalizationProtocol for the EquiPlex Normalization Kit (Multiplex), which normalizes and pools 8 to 96 amplified NGS libraries into an equimolar pool without prior quantification or dilution. The Cas9-based binding workflow preserves double-stranded library integrity and accepts inputs from 5 to 200 nM, with pools eluted ready for direct sequencing on Illumina sequencing platforms.
| EquiPlex Normalization Kit (Singleplex) | EquiPlex Normalization Kit (Multiplex) | |
| Kit contents | Part 1 of 2:
| Part 1 of 2:
|
| Library compatibility |
| |
| Shipping conditions | Ice packs or dry ice | |
| Storage | Part 1 of 2: -20°C ± 5°C Part 2 of 2: 2 - 8°C | |
| Shelf life | (24 rxn): ≥ 6 months (> 24 rxn): ≥12 months | |
What is the EquiPlex™ Normalization Kit?
The EquiPlex Normalization Kit simplifies NGS library preparation prior to short-read sequencing by normalizing and pooling amplified libraries prepared with TruSeq®-style adapters into an equimolar pool, with no prior quantification required. Two kit formats are provided to support the preferred workflow: the EquiPlex Normalization Kit (Singleplex) and the EquiPlex Normalization Kit (Multiplex).
When should I use the EquiPlex Normalization Kit?
The EquiPlex Normalization Kit addresses the challenges of quantifying, normalizing, and pooling amplified libraries prepared with TruSeq®-style adapters ahead of short-read sequencing. The EquiPlex Normalization Kit is ideally suited for libraries made for:
- RNA sequencing, including 3' sequencing
- Methylation sequencing
- DNA sequencing, including ChIP-seq and metagenomics
- Targeted sequencing (post-capture)
- Single cell sequencing (non-tagmentation workflows)
- Amplicon sequencing
Which EquiPlex Normalization Kit should I choose: Singleplex or Multiplex?
The EquiPlex Normalization Kit (Singleplex) produces individually normalized libraries, all at the same concentration, which can then be pooled in equal or variable volumes depending on the desired read allocation for each sample. Libraries remain separate after normalization and individual samples can easily be re-pooled or resequenced if needed.
The EquiPlex Normalization Kit (Multiplex) produces a single pool of normalized libraries in which each library is equally represented in the final pool. This approach minimizes pipetting and sample handling, making it ideal for high-throughput applications where equivalent read counts are desired across all samples.
What adapters are compatible with the EquiPlex Normalization Kit?
The EquiPlex Normalization Kit is designed for amplified libraries prepared with TruSeq-style adapter designs. The compatible adapter design is available in the EquiPlex Normalization Kit User Guides in the Prior to Starting section.
Our Watchmaker Full-Length UDI Adapter Kit and Watchmaker Stubby UDI Adapter Kit are compatible with this kit. Libraries that incorporate Nextera adapter designs are NOT compatible with this kit.
For more detailed guidance on adapter compatibility with the EquiPlex Normalization Kit, please reach out to our support team at support@watchmakergenomics.com or submit a request via our Technical Support form.
Will the EquiPlex Normalization Kit change the size profile of my library or bias fragment size?
The EquiPlex Normalization Kit is designed to preserve the library profile present before normalization and captures a fixed number of library molecules independent of insert size. As a result, the normalization process does not introduce fragment size bias. However, adapter dimers and over-amplified products present in the input library will be carried through the workflow, so library preparation, amplification, and cleanup conditions should be optimized before normalization. Sequencer-specific clustering biases should also be accounted for when pooling normalized libraries with substantially different insert sizes.
What are the required input concentrations for the EquiPlex Normalization Kit?
The EquiPlex Normalization Kit accepts inputs of 10 µL from 5 to 200 nM, and elutes libraries at concentrations ready for direct loading onto short-read sequencing platforms. Workflow modifications for low-concentration libraries with a minimum concentration of 2.5 nM can be found in Appendix A of the EquiPlex Normalization Kit User Guides.
For more detailed guidance on input concentrations, please reach out to our support team at support@watchmakergenomics.com or submit a request via our Technical Support form.
How should I QC my libraries post-normalization?
Post-normalized libraries can be analyzed with various methods defined in Appendix B of the EquiPlex Normalization Kit User Guides. Analysis with Agilent Tapestation ScreenTape® assays, Labchip®, or Bioanalyzer® may need additional dilution or bead-cleanup steps for accurate measurement.
What short-read sequencing platforms is the EquiPlex Normalization Kit compatible with?
Libraries normalized with the EquiPlex Normalization Kit have been demonstrated to be compatible with Illumina® platforms as well as the Element AVITI™ System with Cloudbreak™ Freestyle chemistry.
What plexity can I use when multiplexing libraries with the EquiPlex Normalization Kit?
The multiplex workflow can accommodate any plexity ranging from 8 - 96 libraries. Follow the guidelines in Table 1 of the EquiPlex Normalization Kit (Multiplex) User Guide to plex libraries.
For more detailed guidance on multiplexing libraries, please reach out to our support team at support@watchmakergenomics.com or submit a request via our Technical Support form.
Can the EquiPlex Normalization Kit be automated?
Yes, the EquiPlex Normalization Kits are easily automatable. The kit was developed with automation in mind, including generous overages to account for required dead volumes and stable components. For a comprehensive list of scripts available across popular liquid handling platforms, please visit our automation page. For more information on script accessibility, new script requests, or general automation questions, please reach out to our Automation Team at support@watchmakergenomics.com or submit a request via our Technical Support form.
Where can I go for additional help or technical support?
If you need more detailed guidance or have questions not addressed here, please reach out to our support team at support@watchmakergenomics.com or submit a request via our Technical Support form. Please include details such as product name, lot number, version of protocol you’re using, and a brief description of your question or issue so we can assist you promptly.
Do you offer custom formats?
Yes! Watchmaker offers custom fill volumes, packaging, concentrations, and labeling – including private label – designed to meet your unique needs. We offer flexible terms to serve organizations of any size, and our processes enable rapid turnaround time on customization. Please contact sales@watchmakergenomics.com or submit a request via our Custom Solutions form to learn more about our capabilities.
Are you ISO 13485-certified?
Yes! Our Quality Management System has achieved ISO 13485:2016 Certification. Our certificate has been awarded for the design, development, manufacture, contract manufacture, and support of high performing reagents for genomics applications in medical research. Download the certificate here.
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| Description | 24 rxn | 96 rxn | |
|---|---|---|---|
| EquiPlex Normalization Kit (Singleplex) Incl. reagents for library binding, bead capture, bead washing, and elution | 7BK0016-024 | 7BK0016-096 | Request a quote |
| EquiPlex Normalization Kit (Multiplex) Incl. reagents for library binding, bead capture, bead washing, and elution | 7BK0017-024 | 7BK0017-096 | Request a quote |
Please contact sales@watchmakergenomics.com to inquire about custom kit configurations.
