Dramatic improvements to commercial next generation sequencing (NGS) platforms have resulted in spectacular reductions in the cost-per-base of DNA sequencing. Until relatively recently, the primary focus for innovation has been on the core sequencing technologies, with optimization of library preparation playing a secondary role. The exponential gains in sequencing capacity have simultaneously led to growing sample throughput, increasing the demand for streamlined, efficient, and cost-effective library preparation protocols for multiplexed sample sequencing.
Kapa Biosystems is using novel enzymes engineered via in vitro molecular evolution to develop a suite of innovative products for next generation sequencing library preparation, amplification, and quantification. 

 

1. Library Preparation kits 

KAPA Library Preparation Kits provide all of the enzymes and reaction buffers required for constructing libraries from fragmented dsDNA and include the following modules: End Repair, A-Tailing, Ligation, and Amplification. Reaction buffers are supplied in convenient, concentrated master mix formats comprising all of the required reaction components except oligonucleotide adaptors or PCR primers. 
KAPA Library Preparation Kits are supplied with either a standard PCR library amplification module or a real-time PCR amplification module.

 

2. Library Amplification kits

Available with either a standard PCR library amplification module or a real-time PCR amplification module, KAPA Library Amplification Kits are designed to address PCR-induced bias and improve sequence coverage uniformity. Kits contain the novel KAPA HiFi DNA Polymerase, engineered for high fidelity and processivity and capable of balanced amplification of complex library DNA. KAPA Library Amplification Kits are supplied as the library amplification module contained within KAPA Library Preparation Kits or as a standalone component.

 

KAPA Library Amplification Kit

KAPA Library Amplification Kits are designed for low bias, high fidelity PCR. KAPA HiFi DNA Polymerase was engineered for increased affinity to DNA, without the need for accessory protein domains. The intrinsic high processivity of the enzyme results in significant improvements in yield, sensitivity, speed, target length and the ability to amplify difficult amplicons. These enhancements result in lower amplification bias which leads to more uniform sequence coverage.

 

 

 

 

KAPA Real-Time PCR Library Amplification Kit 

 

KAPA Real-Time PCR Library Amplification Kits are designed to address both enzyme bias and over-amplification. The novel KAPA HiFi DNA Polymerase, engineered for high fidelity and processivity, is capable of balanced amplification of complex library DNA. Real-time monitoring of library amplification provides additional information required to minimize over-amplification. Benefits of performing high fidelity, real-time PCR for next generation sequencing library amplification include:

- Real-time monitoring of amplification allows precise control over the optimal number of PCR cycles.
- Real-time amplification workflows are amenable to automation.
- Real-time amplification plots provide quality metrics for individual enriched libraries, eliminating expensive and time-consuming post-enrichment gel electrophoresis and identifying inconsistencies in library preparation.
- Seamless integration with KAPA Library Quantification Kits.

 

3. Library Quantification kits

Accurate quantification of amplifiable library molecules is critical for the efficient use of next generation sequencing (NGS) platforms – underestimation results in non-clonality, while overestimation leads to inefficiency via poor yields of clonally amplified templates. Accurate library quantification is equally important when pooling indexed libraries for multiplexed sequencing to ensure equal representation of each library. Standard methods for quantifying NGS libraries have a number of important disadvantages. Electrophoresis and spectrophotometry measure total nucleic acid concentrations, whereas optimal cluster density or template-to-bead ratio depends on the appropriate concentration of PCR-amplifiable DNA molecules. These methods also have low sensitivity, consuming nanograms of precious samples, and are not suitable for high-throughput workflows. qPCR is inherently well-suited to NGS library quantification, and overcomes many of the difficulties posed by alternative approaches.