What are the recommended applications for KAPA Library Amplification 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
What type of enzyme is KAPA HiFi HotStart DNA Polymerase?
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.
What are the recommended storage conditions for KAPA HiFi Kits?
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.
What does HotStart formulation mean?
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
What are the recommended applications for 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
What are the recommended applications for 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.
How many cycles should I use to amplify my library?
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.
I am using FFPE samples, how does this affect my amplification protocol?
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.
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 HiFi HotStart 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.
KAPA HiFi HotStart Real-Time Library Amplification Kits
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.