KAPA HiFi Kits

Overview

KAPA HiFi Kits are a family of kits that all contain the same core, engineered KAPA HiFi DNA Polymerase enzyme, formulated for both NGS and PCR applications. KAPA HiFi has become the enzyme of choice for NGS library amplification due to its ability to amplify complex DNA populations with ultra-high fidelity, high efficiency and low amplification bias. This results in lower duplication rates and improved coverage of GC- and AT-rich regions, promoters, low-complexity and other challenging regions. These kits are offered in an easy-to-use ReadyMix or component format, to provide flexibility and convenience.

Benefits of KAPA Library Amplification Kits
  • Higher and more uniform coverage with lower dropout of difficult regions
  • Higher yields, low duplication rates and fewer wasted sequencing reads
  • Higher success rates with different sample types/applications
  • Convenience with the consistency of one core enzyme
  • Trusted as shown through over 10 years in the industry as well as thousands of peer-reviewed publications
     
Product Highlights

Reduced bias and efficient amplification of GC- and
AT-rich genomes

  • Improved representation of all library fragments and sequence regions
  •  Fewer cycles to achieve equivalent yields due to higher amplification efficiency

Improved sequencing coverage

  • Improved coverage uniformity of GC- and AT-rich regions, promoters, and other challenging regions
  •  Increased coverage depth of difficult regions in targeted capture workflows, where two amplification steps are performed

Amplification of NGS libraries with ultra-high fidelity

  • Enhanced proofreading (3′-5′ exonuclease) activity using KAPA HiFi enzyme
  • Ultra-high fidelity for NGS applications

*Data on file.

Research Use Only. Not for use in diagnostic procedures.
KAPA is a trademark of Roche. Other product names and trademarks are the property of their respective owners

Roche Sequencing Solutions offers a family of KAPA HiFi Kits, containing the same core, engineered KAPA HiFi DNA Polymerase enzyme, formulated for both NGS and PCR applications. These kits are available in different formats and formulations, to provide flexibility and convenience across a broad range of applications.

The table below describes the specifications for the KAPA HiFi Kits:

  KAPA HiFi PCR Kit   KAPA HiFi HotStart PCR Kit KAPA HiFI HotStart ReadyMix PCR Kit KAPA HiFi HotStart Library Amplification Kit KAPA HiFi HotStart Library Amplification Kit with Primer Mix KAPA HiFi HotStart Real-Time Library Amplification Kit
HotStart Enzyme No Yes Yes Yes Yes Yes
Format Components Components ReadyMix ReadyMix ReadyMix with Primer Mix ReadyMix
Kit Contents KAPA HiFi enzyme
KAPA HiFi buffers
Magnesium Chloride
dNTPs
KAPA HiFi HotStart enzyme
KAPA HiFi buffers
Magnesium Chloride
dNTPs
KAPA HiFi HotStart ReadyMix KAPA HiFi HotStart ReadyMix
KAPA HiFi HotStart ReadyMix
Amplification Primer Mix
KAPA HiFi HotStart ReadyMix
Fluorescein Standards
Recommended
Applications
PCR applications requiring flexibility and/or optimization PCR applications requiring a HotStart enzyme and flexibility and/or optimization Routine use in established PCR and NGS assays Routine NGS library amplification on any platform using user-supplied primers Routine NGS library amplification for Illumina® platforms NGS library amplification for Illumina® platforms that require careful control

Research Use Only. Not for use in diagnostic procedures.
KAPA is a trademark of Roche. Other product names and trademarks are the property of their respective owners

Research Use Only. Not for use in diagnostic procedures.
KAPA is a trademark of Roche. Other product names and trademarks are the property of their respective owners

Kit Code Roche Cat. No
Description
Kit Size
How to buy

KK2103

07958854001

KAPA HiFi PCR Kit (20 U)

20 x 50 µL reactions

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KK2101

07958838001

KAPA HiFi PCR Kit (100 U)

100 x 50 µL reactions

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KK2102

07958846001

KAPA HiFi PCR Kit (250 U)

250 x 50 µL reactions

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KK2500

07958862001

KAPA HiFi HotStart PCR Kit (20 U)

20 x 50 µL reactions

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KK2501

07958889001

KAPA HiFi HotStart PCR Kit (100 U)

100 x 50 µL reactions

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KK2502

07958897001

KAPA HiFi HotStart PCR Kit (250 U)

250 x 50 µL reactions

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KK2600

07958919001

KAPA HiFi HotStart ReadyMix PCR Kit (0.25 mL Sample)

10 x 50 µL reactions

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KK2601

07958927001

KAPA HiFi HotStart ReadyMix PCR Kit (1.25 mL)

50 x 50 µL reactions

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KK2602

07958935001

KAPA HiFi HotStart ReadyMix PCR Kit (6.25 mL)

250 x 50 µL reactions

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KK2610

07958943001

KAPA HiFi HotStart Library Amplification Kit (10 rxn)

10 x 50 µL reactions

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KK2611

07958951001

KAPA HiFi HotStart Library Amplification Kit (50 rxn)

50 x 50 µL reactions

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KK2612

07958960001

KAPA HiFi HotStart Library Amplification Kit (250 rxn)

250 x 50 µL reactions

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KK2620

07958978001

KAPA HiFi HotStart Library Amplification Kit with Primer Mix (50 rxn)

50 x 50 µL reactions

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KK2621

07958986001

KAPA HiFi HotStart Library Amplification Kit with Primer Mix (250 rxn)

250 x 50 µL reactions

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KK2623

07958994001

KAPA Library Amplification Primer Mix Kit (250 rxn)

250 x 50 µL reactions

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KK2702

07959028001

KAPA HiFi HotStart Real-Time Library Amplification Kit (250 rxn)

250 x 50 µL reactions

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KK2709

07959036001

KAPA HiFi HotStart Real-Time Library Amplification Standards Kit (4 x 1.5 mL)

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Research Use Only. Not for use in diagnostic procedures.
KAPA is a trademark of Roche. Other product names and trademarks are the property of their respective owners

 

All KAPA HiFi Kits

KAPA HiFi Kits are a family of kits that all contain the same core KAPA HiFi DNA Polymerase enzyme, engineered using directed evolution technology. These kits are available in different formats to provide flexibility for both NGS and PCR applications, including:

  • KAPA HiFi PCR Kits 
  • KAPA HiFi HotStart PCR Kits 
  • KAPA HiFi HotStart ReadyMix PCR Kits 
  • KAPA HiFi HotStart Library Amplification Kits 
  • KAPA HiFi HotStart Library Amplification Kits with Primer Mix 
  • KAPA HiFi HotStart Real-Time Library Amplification Kits

KAPA HiFi HotStart DNA Polymerase is a novel B-family DNA polymerase exhibiting performance in comparison with other high-fidelity DNA polymerases and polymerase blends. The enzyme was engineered for high efficiency, low-bias and high fidelity amplification. This leads to an increased number of samples processed successfully, allowing researchers to obtain more information out of every sample and maximize sequencing economy.

Upon receipt, store the entire kit at -20°C in a constant-temperature freezer. Kits may be stored at 4°C for regular, short-term use (up to one month). Provided that it has been handled carefully and not contaminated, the product is not expected to be compromised if left at room temperature for short periods of time (up to three days). Long-term storage at room temperature or 4°C is not recommended. Please note that reagents stored above -20°C are more prone to degradation when contaminated by the user; storage at such temperatures is therefore at the user’s own risk.

KAPA HiFi HotStart Real-Time Library Amplification Kits contain fluorescent standards, which should be protected from direct light. Exposure to direct light for an extended period of time may result in loss of fluorescent signal intensity.

 

A proprietary antibody inactivates the polymerase until the first cycle of thermal denaturation. This minimizes spurious amplification products that may result from non-specific priming events during reaction setup and initiation and increases overall reaction efficiency.

 

KAPA HiFi PCR Kits, KAPA HiFi HotStart PCR Kits and KAPA HiFi HotStart ReadyMix PCR Kits

KAPA HiFi PCR Kits and KAPA HiFi HotStart PCR Kits are provided in a component format to allow flexibility and optimization for a range of PCR applications in both PCR and NGS workflows. These kits contain all the components required for amplification, including the KAPA HiFi enzyme (either non-HotStart or HotStart), KAPA HiFi buffers, MgCl2 and dNTPs.

KAPA HiFi HotStart ReadyMix PCR Kits contain KAPA HiFi HotStart DNA Polymerase in a ready-to-use master mix format, for routine use in established PCR and NGS workflows. 

 

 

KAPA HiFi HotStart Library Amplification Kits

KAPA HiFi HotStart Library Amplification Kits are ideally suited for the amplification of NGS libraries carrying appropriate adapter sequences and the enrichment of targeted capture sequences where high efficiency, low-bias and high-fidelity amplification is required.

The optimal cycle number is determined by the volume and concentration of adapter-ligated, purified library DNA added to each enrichment PCR reaction. Typically, this is in the range of 5 - 18 cycles but may require optimization depending on workflow and sequencing instrument. If cycled to completion (not recommended), a single library amplification PCR can produce 8 – 10 µg (160 – 200 ng/µL) of amplified library. To minimize over-amplification, the number of amplification cycles should be optimized to produce an amplified library with a concentration in the range of 10 – 30 ng/µL.

Quantification of adapter-ligated libraries prior to library amplification can greatly facilitate the optimization of library amplification parameters, particular when a library construction workflow is first established. By using the KAPA Library Quantification Kit, the amount of adapter-ligated molecules available for library amplification can be determined accurately. From this, the number of amplification cycles needed to achieve a specific yield of amplified library may be predicted.

 

The optimal number of amplification cycles may be 1 – 3 cycles higher for libraries constructed from FFPE DNA or other challenging samples, or libraries with a broad fragment size distribution.
 

These higher molecular weight peaks are artifacts of over-amplification. In library amplification reactions, primers are typically depleted before dNTPs. When DNA synthesis can no longer take place due to primer depletion, subsequent rounds of DNA denaturation and annealing result in the separation of complementary DNA strands, followed by imperfect annealing to non-complementary partners by way of the adapter sequences. This presumably results in the formation of long, mostly single-stranded, so-called “daisy-chains”, comprising large assemblies of improperly annealed, partially double-stranded, heteroduplex DNA.

In most cases the “daisy-chained” molecules are bona fide library molecules that are temporarily annealed to one another to form longer concatemers. Since these heteroduplexes contain significant portions of single-stranded DNA, over-amplification leads to the under-quantification of library molecules with assays employing dsDNA-binding dyes. qPCR-based library quantification methods, such as the KAPA Library Quantification assay, quantify DNA by denaturation and amplification, thereby providing an accurate measure of the amount of adapter-ligated molecules in a library, even if the library was over-amplified.

Excessive library amplification can result in unwanted artifacts such as PCR duplicates, chimeric library inserts, and nucleotide substitutions. The extent of library amplification should therefore be limited as much as possible, while ensuring that sufficient material is generated for QC and downstream processing (sequencing or target enrichment).
 

Yes, this kit is compatible with the Nextera® Sample Preparation protocol. Use 98°C as the denaturation temperature.

 

KAPA HiFi HotStart Real-Time Library Amplification Kits

  • 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 time-consuming post-enrichment gel electrophoresis and identifying inconsistencies in library preparation
  • Seamless integration with KAPA Library Quantification Kits

The four fluorescent standards supplied with the kit will provide a range for when to terminate amplification. Optimal amplification for NGS applications corresponds to the region between fluorescent standard 1 and 3. The termination cycle number should be adjusted accordingly without the requirement for performing gel electrophoresis. Each plate must contain a set of fluorescent standards (each loaded in triplicate).

  • If the linear amplification profile of the library is significantly below fluorescent standard 1 at the end of qPCR cycling, then it is unlikely that there will be sufficient library material to sequence after PCR purification.
  • If the linear amplification profile of the library is significantly above fluorescent standard 3 at the end of the qPCR cycling, then the library has been over-amplified. This may lead to amplification bias, higher error rates, and/or the presence of chimeric PCR products.
     

Yes, the amplification plots can be used in real-time to select the optimal cycle without a pre-programmed termination cycle. To do this, program 30 cycles into the real-time thermocycler. After starting the real-time thermocycler, wait until the desired fluorescence of the library is achieved before terminating the real-time reaction. Please note that it is critical to terminate the reaction directly after data acquisition at 72°C and before the tube ramps to 95°C for the start of the next cycle. This will ensure that the enriched library DNA remains double-stranded for effective downstream purification.

Research Use Only. Not for use in diagnostic procedures.
KAPA is a trademark of Roche. Other product names and trademarks are the property of their respective owners