KAPA Library Amplification Kits

Overview

KAPA Library Amplification Kits include KAPA HiFi DNA Polymerase, a novel enzyme engineered using our directed evolution technology for ultra-high fidelity and robustness. KAPA HiFi has become the enzyme of choice for next-generation sequencing (NGS) library amplification due to its ability to amplify complex DNA populations with high fidelity, high efficiency and very 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.

Benefits of KAPA Library Amplification Kits

  •  Low amplification bias
  •  Improved sequencing coverage uniformity of difficult regions
  •  Industry-leading fidelity

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 industry-leading fidelity*

  • Enhanced proofreading (3′-5′ exonuclease) activity using KAPA HiFi enzyme
  • Industry-leading fidelity confirmed by pyrosequencing

*Data on file.

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.

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
Components
  Kit Code   2XKAPA HiFi HotStart ReadyMix 2X KAPA HiFI HotStart Real time PCR Master Mix Fluorescein Primers
Standard Kit

KK2611

KK2612

                  X      
With primer mix

KK2620

KK2621

                   X           X
Real time PCR lbrary amplification with fluoroscent standards

KK2701

KK2702

 

                                   

                                  X

         

       X

 

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

The KAPA Library Amplification Kit is ideally suited for the amplification of libraries carrying appropriate adapter sequences and the enrichment of targeted capture sequences where:

  • low amplification bias is required in order to maintain genome representation, or
  • high fidelity is required.

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.

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.

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.

 

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.

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 ReadyMix 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.

  • 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.

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

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). 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. Minimize exposure of the Master Mix (2X) and fluorescent standards 1-4 to direct light. Exposure to direct light for an extended period of time may result in loss of fluorescent signal intensity.

 

 

Real-Time Library Amplification Kits FAQs

  • 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.

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

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). 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. Minimize exposure of the Master Mix (2X) and fluorescent standards 1-4 to direct light. Exposure to direct light for an extended period of time may result in loss of fluorescent signal intensity.