Amplify without compromise
Polymerase slippage across highly repetitive and short tandem repeat regions makes accurate library amplification challenging, driving indel errors that can obscure true biological signal.
Equinox® Prime Library Amplification Kits are engineered to address this issue, lowering indel and base misincorporation error rates without compromising performance in other critical areas such as yield, GC coverage uniformity, amplification efficiency, and overall fidelity. This enables accurate and consistent variant detection across demanding applications including microsatellite instability (MSI) and minimal residual disease (MRD) assay development, whole genome sequencing, and targeted panels.
Key Features and Benefits
- Reduced polymerase slippage across homopolymer regions improves indel accuracy for stringent applications, such as MSI profiling, whole-genome, and targeted sequencing
- High-fidelity amplification reduces PCR errors and enables sensitive variant calling
- Highly uniform GC coverage improves sequencing economy and ensures challenging genomic regions can be interrogated
- Efficient amplification enables users to minimize PCR cycles to achieve desired yields
- Compatible with paramagnetic bead-based hybridization capture workflows for both somatic and germline testing applications
Figure 1. Reduced polymerase slippage improves variant calling accuracy
Applications
- Somatic oncology research
- Oncology assay development for the analysis of:
- Microsatellite Instability (MSI)
- Minimal Residual Disease (MRD)
- Homologous Recombination Deficiency (HRD)
- Tumor Mutation Burden (TMB)
- Whole genome sequencing (WGS)
- Whole exome sequencing
- Targeted sequencing protocols employing hybridization capture
- Tumor profiling, including FFPE
- Liquid biopsy (cfDNA/ctDNA) studies
- Low-frequency variant detection
- Early detection and high-sensitivity screening assays
Engineered without trade-offs
Library amplification has historically required users to make performance trade-offs, such as high yield and GC coverage uniformity at the expense of fidelity and slippage. Equinox Prime was engineered to overcome those limitations, delivering unmatched performance across every metric that determines data quality and sequencing economy.
Figure 2. Equinox Prime achieves top-tier performance ... MORE
Why each metric matters
- Low PCR Error Rates
Reduce false positives for more confident detection of low-frequency variants. - Reduced Polymerase Slippage
Improves indel calling accuracy and ensures reliable detection in repeat and homopolymer regions. - High Yield Per Cycle
Minimizes the number of PCR cycles needed, reducing amplification bias and PCR errors. - Terminal Yield
Supports input requirements for downstream workflows such as hybrid capture, without compromising data quality. - Even GC Coverage
Reduces dropouts and over-representation, improving sequencing economy.
Engineered without trade-offs
Figure 2. Equinox Prime achieves top-tier performance across all five library amplification metrics. Values are normalized so that the outermost boundary represents best-in-class performance for each metric, with larger area indicating better overall performance and ideal being a perfect pentagon. The same human whole genome library was amplified with each polymerase following manufacturer’s recommendations.
Key Performance Data
High-fidelity amplification for sensitive variant detection
Polymerase base substitution errors accumulate with each PCR cycle and can be mistaken for true low-frequency variants in sensitive applications. Equinox Prime achieves the lowest total error rate among leading library amplification reagents. The reduction in C>T misincorporation is an especially relevant measure for oncology applications, where this substitution is among the most common somatic mutation types.
High-fidelity amplification for sensitive variant detection
Minimize slippage artifacts for more accurate indel calling
Homopolymer regions and other highly repetitive loci are among the most challenging sequences to amplify accurately. Polymerase slippage through these regions generates false indels that inflate artifact rates and undermine variant calling confidence, posing a significant challenge in applications such as MSI profiling. Equinox Prime is engineered to maintain high fidelity through difficult repeat regions, producing more accurate libraries with improved indel calling.
Minimize slippage artifacts for more accurate indel calling
Uniform GC coverage delivers efficient sequencing
Amplification bias can skew sequence coverage, leading to gaps or over-representation that reduce data quality and increase sequencing costs. Equinox Prime maintains consistent normalized coverage across a range of GC content. This uniform coverage extends to challenging, GC-rich loci such as IGSF8, a gene implicated in tumor immune evasion. Sequence dropout can leave these regions underrepresented or unresolved.
Uniform GC coverage delivers efficient sequencing
Higher yields and on-bead amplification support hybrid capture workflows
Equinox Prime generates higher library yields than competing amplification mixes, reaching target amounts with fewer PCR cycles and limiting the accumulation of associated errors and bias. This efficiency advantage is particularly valuable in hybridization capture workflows that often require high library mass. These workflows also typically involve amplification directly on streptavidin beads, placing additional demands on polymerase performance. Equinox Prime maintains low-bias, uniform amplification in the presence of paramagnetic beads, supporting consistent coverage across target loci and reducing the risk of sequence dropout.
Higher yields and on-bead amplification support hybrid capture workflows
BROCHURES
Equinox Prime Library Amplification Kits Brochure
Element Illumina Singular Ultima DNA NGS RNA NGS Equinox Prime Library AmplificationEquinox® Prime Library Amplification Kits deliver high-fidelity amplification without compromising performance on yield, GC coverage uniformity, or PCR efficiency, supporting accurate variant detection across demanding NGS applications.
PROTOCOLS
Equinox Prime Library Amplification Kit User Guide
Element Illumina Singular Ultima DNA NGS RNA NGS Equinox Prime Library AmplificationProtocol for the Equinox® Prime Library Amplification Kit, which delivers high-fidelity amplification without compromising performance on yield, GC coverage uniformity, or PCR efficiency, supporting accurate variant detection across demanding NGS applications.
| Kit contents | Equinox® Prime Amplification Master Mix (2X) |
|---|---|
| Concentration | 2X |
| Shipping conditions | Ice packs |
| Storage | Longterm: -20°C ± 5°C Short term: 4°C for up to 2 weeks |
| Stability | 20 freeze-thaw cycles |
| Shelf life | (24 rxn): ≥ 6 months (> 24 rxn): ≥ 12 months |
Is Equinox Prime replacing Equinox Library Amplification Kits?
No, Equinox Prime is not replacing Equinox Library Amplification Kits. Equinox will continue to be available for purchase. Equinox Prime works across a broad range of applications and was developed for oncology and highly sensitive applications where high fidelity is critical for accurate single nucleotide variant (SNV) and indel characterization.
How is Equinox Prime different from Equinox Library Amplification Kits?
Equinox Prime has improved fidelity and reduced slippage across homopolymer repeat-rich regions such as those used in MSI testing. Both mixes maintain excellent GC coverage, bead compatibility, and yield.
What are the relevant applications for the Equinox Prime Amplification Kit?
- Efficient library amplification from a wide range of template amounts (0.1 pg – 500 ng) up to 1 kb in length
- Low-frequency variant detection NGS assays, including those utilizing challenging samples such as FFPE and cfDNA
- Hybridization capture workflows
- Single-cell analysis
- Whole genome sequencing (WGS)
- Amplicon sequencing
- Indexing PCR
- RNA-Seq
- ChIP-Seq, ATAC-Seq, and associated epigenetic applications
- Illumina® and non-Illumina sample preparation workflows
What type of enzyme is in the Equinox Prime Library Amplification Kit?
Equinox Prime DNA Polymerase is an engineered B family proofreading polymerase variant developed specifically for NGS applications to deliver excellent fidelity, minimal slippage, uniform sequence coverage, and high library complexity.
Are reaction buffer and dNTPs supplied in the Equinox Prime Library Amplification Kit?
Yes! The Equinox Prime Amplification Master Mix consists of an optimized PCR buffer, hot start enzyme formulation, and dNTPs.
Can the Equinox Prime Library Amplification Kit amplify long templates?
Equinox Prime Library Amplification Kits can amplify templates up to 9 kb in length. Amplification beyond 9 kb is possible but may require additional optimization.
Does the Equinox Prime Library Amplification Kit have hot start functionality?
Yes, the Equinox Prime Library Amplification Kit is supplied in a hot start format. A highly optimized hot start antibody is used to inhibit polymerase and exonuclease activity prior to the initial denaturation (enzyme activation) step. Equinox Prime DNA Polymerase exhibits industry-leading inhibition of both polymerase and exonuclease activity prior to activation.
Do Equinox Prime Library Amplification reagents have exonuclease activity?
Equinox Prime Library Amplification reagents exhibit 3' to 5' exonuclease activity, commonly referred to as proofreading activity. Hot start functionality inhibits this activity prior to heat activation. To prevent 3’ primer degradation, we recommend using primers that have phosphorothioate bonds at one or more sites.
Equinox Prime Library Amplification reagents do not exhibit 5' to 3' exonuclease activity.
What are the cycling recommendations for the Equinox Prime Library Amplification Kit?
Please reference our User Guide for recommendations on Library Amplification protocol instructions on reaction setup conditions and cycling protocol.
What primer concentrations are recommended for amplification with the Equinox Prime Library Amplification Kit?
Optimal primer concentration depends on the application and should be optimized accordingly. Forward and reverse primers should always be present in equivalent concentrations, with final primer pair concentrations ranging from 1 to 2 µM each. A final primer pair concentration of 1 µM is sufficient to yield 500 ng of library (approximately 40 nM in a 50 µL reaction for a library with an average fragment length of 400 bp). If the total library yield must exceed 500 ng or 40 nM, use a final concentration of 2 µM.
Please refer to the User Guide for primer concentration recommendations. If you need more specific recommendations for primers, please contact support@watchmakergenomics.com or submit a request via our Technical Support form.
Are protected primers required for Equinox Prime library amplification-based PCR amplification?
Yes, protection of the 3'-ends of amplification primers through the incorporation of one or more phosphorothioate bonds is highly recommended to achieve optimal specificity and library yields.
What is the optimal annealing temperature for the Equinox Prime Library Amplification Kit?
The optimal annealing temperature depends on the adapter format used. For the Watchmaker Full-Length UDI Adapter Kit with the standard Illumina "P5" and "P7" primers an annealing temperature of 60°C is recommended. For Stubby UDI Adapters an annealing temperature of 55°C is recommended.
For alternatively-supplied primers, please contact support@watchmakergenomics.com or submit a request via our Technical Support form.
What are the recommended extension times for amplification with the Equinox Prime Library Amplification Kit?
A 30 sec extension is sufficient for libraries with a mode fragment size up to 500 bp. A 45 sec extension time is recommended for libraries with mode fragment sizes >500 bp.
Is the Equinox Prime Library Amplification Kit compatible with amplification on SPRI beads?
Yes, the Equinox Prime library amplification kits are compatible with up to 100 µL of SPRI beads. See Appendix A of the User Guide for more specifics or contact support@watchmakergenomics.com or submit a request via our Technical Support form for additional guidance.
Is the Equinox Prime Library Amplification Kit compatible with amplification on Streptavidin beads or beads commonly used in hybridization capture workflows?
Yes, the Equinox Prime library amplification kits are compatible with up to 500 µg of various types of beads used in hybridization capture workflows. See Appendix A of the User Guide for more specifics or contact support@watchmakergenomics.com or submit a request via our Technical Support form for additional guidance.
Which library preparation kits is the Equinox Prime Library Amplification Kit compatible with?
Equinox Prime library amplification kits are compatible with all of the Watchmaker DNA and RNA Library Preparation Kits. For specific guidance please contact support@watchmakergenomics.com or submit a request via our Technical Support form for additional guidance.
Do you offer a 4X master mix of Equinox Prime?
No, Equinox Prime is only available in a 2X master mix format. If a 4X master mix is required for your application then we recommend our Equinox High Concentration Library Amplification Kit.
Do you offer a uracil-tolerant library amplification mix?
Yes, please see our Equinox Uracil Tolerant Library Amplification Kit.
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 | 384 rxn | |
|---|---|---|---|---|
| Equinox® Prime Library Amplification Kit incl. P5/P7 Primer Mix (10x) | 7K0141-024 | 7K0141-096 | 7K0141-384 | Request a quote |
| Equinox® Prime Library Amplification Kit (w/o primers) | 7K0140-024 | 7K0140-096 | 7K0140-384 | Request a quote |
Please contact sales@watchmakergenomics.com to inquire about custom kit configurations.
