KAPA Library Amplification Kits

The gold standard for NGS library amplification.

KAPA Library Amplification Kits contain KAPA HiFi DNA Polymerase, a novel enzyme engineered for ultra-high fidelity and robustness using Kapa’s directed evolution technology platform.

KAPA HiFi has become the enzyme of choice for NGS library amplification due to its ability to amplify complex DNA populations with high fidelity, high efficiency, decreased PCR duplication rates and very low bias.* This results in lower duplication rates and improved coverage of GC- and AT rich regions, promoters, low complexity and other challenging regions in all NGS library construction workflows requiring library amplification.

In addition to the standard library amplification formulation, KAPA HiFi is available in a unique real-time formulation, for applications that demand precise control over library amplification. A uracil-tolerant variant, KAPA HiFi Uracil+ is also available for the high-efficiency, high-fidelity, low-bias amplification of libraries constructed from bisulfite-treated DNA.

KAPA Library Amplification Kits for Illumina® platforms now also include an optimally formulated Library Amplification Primer Mix.

*Data on file.

For Research Use Only. Not for use in diagnostic procedures.

Product Highlights

Achieve the lowest amplification bias and duplication rates

  • Lower amplification bias result in improved representation of all library fragments and sequence regions
  • High-efficiency amplification leads to fewer amplification cycles and lower PCR duplicates
  • Less additional next-generation or Sanger sequencing needed to complete genomes
The first exons of many genes have a high GC content, and are therefore difficult to amplify. Regions of these exons are therefore typically underrepresented in whole exome sequence data. Pre-capture amplification with the evolved, low-bias KAPA HiFi DNA Polymerase results in significantly improved coverage of GC- and AT-rich regions, and other sequence elements that are difficult to amplify. Data courtesy of The Broad Institute. Data on file.

Improve coverage of GC- and AT-rich regions

  • Lower amplification bias improves coverage uniformity of GC- and AT-rich regions, promoters, low complexity and other challenging regions
  • Improved overall coverage and coverage uniformity observed on both Illumina and Ion Torrent™ sequencing platforms
Real-time, high fidelity amplification of multiple libraries with the KAPA Real-Time Library Amplification Kit. Twenty libraries, spanning a ~64-fold concentration range (equivalent to 6 cycles), were simultaneously amplified and terminated after 14 cycles. Fourteen of the twenty libraries fall within the targeted amplification range (the fluorescence range defined by the Real-Time Amplification Standards 1¬–3). The remaining six libraries could either be used as is, noting that they may be outside the optimal concentration range, or they could be re-amplified individually or in high- or low-concentration groups. Data on file.

Exercise precise control over library amplification

  • KAPA Real-Time Amplification Kits allow for library amplification to be observed in real time
  • Terminate amplification at the optimal point for individual samples
  • Optimize library amplification parameters for higher throughput workflows
Error rates of Taq DNA polymerase and polymerases typically used in NGS library amplification. The error rates of proofreading DNA polymerases such as Q5 (New England Biolabs), Phusion (Thermo Scientific) and the evolved KAPA HiFi DNA Polymerase is 50 – 100 times lower than that of Taq. The error rate of KAPA HiFi was confirmed by deep sequencing on the 454 platform, and is 1 error per 3.54 x 106 nucleotides incorporated. Data on file.

Amplify NGS libraries with industry-leading fidelity

  • KAPA HiFi was engineered using directed evolution to have enhanced proofreading (3′-5′ exonuclease) activity
  • Industry-leading fidelity confirmed by 454 sequencing
Applications
  • Amplification of Illumina libraries (DNA and RNA sequencing)
  • Real-time amplification of libraries prepared from low input, ChIP DNA and other precious samples
  • Pre- and post-capture amplification in targeted capture workflows
Kit Specifications and Contents / Storage

Kits can be stored for up to 12 months at -20˚C.

Kits include KAPA HiFi HotStart ReadyMix (2X), a convenient PCR master mix containing KAPA HiFi HotStart DNA Polymerase, KAPA dNTPs, reaction buffer, and MgCl2 at a final concentration of 2.5 mM. Kits with primers are also available.

Kits for real-time library amplification include a real-time version of the KAPA HiFi HotStart ReadyMix, as well as fluorescent standards to monitor library amplification.

Components

LAK_Component Chart

Specifications

Spec
Description
Compatible Platform
Illumina HiSeq, MiSeq, NextSeq, and GAIIx
Library Type
DNA, RNA, and bisulfite-treated DNA
Starting Material
Any NGS library that requires amplification
Sequencing Applications
Whole Genome Sequencing Whole Exome Sequencing Targeted Sequencing (custom panels) RNA-Seq ChIP-Seq Amplicon Sequencing
FAQs
Which enzyme is used in the KAPA Library Amplification Kits?

The KAPA Library Amplification Kit contains KAPA HiFi HotStart DNA Polymerase in a ready-to-use master mix format. It is an antibody-based hot-start formulation of KAPA HiFi DNA Polymerase, a novel B-family DNA polymerase exhibiting industry-leading performance in comparison with other high-fidelity DNA polymerases and polymerase blends. The enzyme was engineered for increased affinity to DNA. This 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.

How many cycles should I use to amplify my library?

The optimal cycle number is determined by the volume and concentration of adapter-ligated, size-separated purified library DNA added to each enrichment PCR reaction. Typically this is in the 5-18 cycle range but may require optimization depending on workflow. 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.

I am using FFPE samples, how does this affect my amplification protocol?

The actual 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.

What is the secondary, higher molecular weight peak observed in the Bioanalyzer trace of my amplified library?

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.

How does over-amplification affect sequencing of my library?

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).

Is the KAPA Library Amplification Kit compatible with the Nextera Sample Preparation protocol?

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

 

Real-Time Library Amplification Kits FAQs

What are the benefits of performing high-fidelity, real-time PCR for next-generation sequencing library amplification?

  • 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

How many cycles should I use to amplify my library?

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).

Why is it important to terminate the amplification reaction between fluorescent standard 1 and 3?

  • 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.

Can I terminate the reaction by monitoring the raw data linear real-time amplification plots in real time?

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.

Is the KAPA Real-Time Library Amplification Kit compatible with the Nextera® Sample Preparation protocol?

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

Ordering

Kits include KAPA HiFi HotStart ReadyMix (2X), a convenient PCR master mix containing KAPA HiFi HotStart DNA Polymerase, KAPA dNTPs, reaction buffer, and MgCl2 at a final concentration of 2.5 mM. Kits with primers are also available. Kits for real-time library amplification include a real-time version of the KAPA HiFi HotStart ReadyMix, as well as fluorescent standards to monitor library amplification.

Kit Code
Roche Cat. No
Description
Kit Size
How to buy
KK2611
07958951001
Standard kit
50 x 50 µL reactions
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KK2612
07958960001
Standard kit
250 x 50 µL reactions
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KK2620
07958978001
With primer mix
50 x 50 µL reactions
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KK2621
07958986001
With primer mix
250 x 50 µL reactions
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KK2702
07959028001
Real-time PCR library amplification, with fluorescent standards
250 x 50 µL reactions
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