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适用说明:本说明书适用于 KAPA Real-time Library Amplification Kit (07959028001), 及KAPA Real-time Library Amplification Standards Kit (07959036001).
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Product Description

High-fidelity PCR is used to selectively enrich library fragments carrying appropriate adapter sequences and to amplify the amount of DNA prior to sequencing. During PCR enrichment of libraries, minimizing amplification bias is critical to ensure uniform sequence coverage. Amplification bias occurs when a DNA polymerase is unable to amplify all targets within a complex population of library DNA with equal efficiency. Bias is further exacerbated when libraries are over-amplified.

KAPA Real-time Library Amplification Kits are designed to address both sources of PCR-induced bias. 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.

KAPA Real-time Library Amplification Kits contain KAPA HiFi HotStart Real-time PCR Master Mix (2X), a ready-to-use cocktail containing all components for PCR, except primers and template. The master mix contains KAPA HiFi HotStart DNA Polymerase in a proprietary reaction buffer, dNTPs, MgCl2 (2.5 mM at 1X), SYBR® Green I dye and stabilizers. Four fluorescent standards are supplied, and are used to define a window for optimal amplification (Figures 1 and 2).

KAPA HiFi HotStart DNA Polymerase 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 (B-family) DNA polymerases and polymerase blends. 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. In the HotStart formulation, a proprietary antibody inactivates the polymerase until the first denaturation step. This eliminates spurious amplification products resulting from non-specific priming events during reaction setup and initiation and increases overall reaction efficiency.

KAPA HiFi HotStart DNA Polymerase has 5'→3' polymerase and 3'→5' exonuclease (proofreading) activities, but no 5'→3' exonuclease activity. The strong 3'→5' exonuclease activity results in superior accuracy during DNA amplification. The error rate of KAPA HiFi HotStart DNA Polymerase is ~2.8 x 10-7. This fidelity is approximately 100X higher than that of wild-type Taq and up to 30X higher than polymerase blends. The presence of SYBR Green I dye in the reaction does not compromise fidelity. DNA fragments generated with KAPA HiFi HotStart Real-time PCR Master Mix (2X) may be used for routine downstream applications, including restriction enzyme digestion and sequencing. PCR products generated with KAPA HiFi HotStart Real-time PCR Master Mix (2X) are blunt-ended, but may be 3'-dA-tailed for cloning into TA cloning vectors.

Library Application Protocol

  1. Reagent Preparation
    1.1
    Thaw the primers (sold separately) required for PCR enrichment, a tube of KAPA HiFi HotStart Real-time PCR Master Mix (2X) and Fluorescent Standards 1 – 4 at room temperature. 
    Note: KAPA HiFi HotStart Real-time PCR Master Mix (2X) thaws easily, however, due to the high viscosity it is important to vortex well before use. The Fluorescent Standards should be thawed for at least 15 min before use.
    1.2
    Mix and briefly centrifuge the thawed KAPA HiFi HotStart Real-time PCR Master Mix (2X), primer,and Fluorescent Standards 1 – 4.
    1.3
    Thaw and briefly centrifuge the adapter-ligated, size-separated, purified library DNA.
    1.4
    Pre-program the real-time thermocycler. 

  2. Reaction Setup
    Each plate must contain a set of Fluorescent Standards 1 – 4 (each loaded in triplicate) in addition to a single 50 μL real-time PCR reaction for each library requiring amplification.

    In order to maintain optimal library diversity it is necessary to add sufficient adapter-ligated library DNA to each enrichment PCR reaction. The optimal cycle number is dependent on the volume and concentration of library material added to each 50 μL PCR reaction. High background fluorescence may result if >100 ng dsDNA template is added per 50 μL real-time PCR reaction. To ensure accurate results avoid; overfilling of the wells, bubbles in reactions, or anything else that could distort the fluorescent signal.
    2.1
    Assemble each library amplification reaction as follows:

    * Or another suitable 10X library amplification primer mix. The recommended final concentration of each primer in the library amplification reaction is 0.5 – 2 μM.

    2.2
    Add 50 μL of each fluorescent standard in triplicate to wells of the real-time PCR plate.
    2.3
    Seal the plate, mix gently, and centrifuge briefly.

  3. Cycling Protocol
    • If conventional end-point PCR has previously been used successfully and the same amount and type of library is added to the KAPA HiFi HotStart Real-time PCR reactions, then program the real-time thermocycler with the same number of cycles as previously used.
    • It is important to ensure that data acquisition is performed at 72 °C. Amplify using the following cycling protocol:

    * Optimization of the annealing temperature may be required for non-standard (i.e., other than Illumina TruSeq®) adapter/primer combinations.
    * The optimal cycling number will depend upon the volume and concentration of adapter-ligated, size separated, purified library DNA added to each enrichment PCR reaction. Typically, this is in the 10 – 18 cycle range but may require optimization.

  4. Data Analysis and Interpretation
    Initially, the raw data (i.e., not background subtracted) linear real-time amplification plots can be used as a built-in quality metric to validate the level of amplification of each amplified library.
    • 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 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.
    This data is also useful as a quality control metric for identifying inconsistencies during library preparation between multiple libraries.

    Note: the amplification plots can also be used in real-time to select the optimal cycle without a preprogrammed 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.

    Note: it is critical to terminate the reaction directly after data acquisition at 72 °C and before the tube ramps to 95º C for the start of the next cycle. This will ensure that the enriched library DNA remains double-stranded for efficient downstream purification.

  5. Bead-based Cleanup
    5.1
    In the library amplification plate/tube(s), perform a 1X bead-based cleanup by combining the following:


    5.2
    Mix thoroughly by vortexing and/or pipetting up and down multiple times.
    5.3
    Incubate the plate/tube(s) at room temperature for 5 – 15 min to bind DNA to the beads.
    5.4
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    5.5
    Carefully remove and discard the supernatant.
    5.6
    Keeping the plate/tube(s) on the magnet, add 200 μL of 80% ethanol.
    5.7
    Incubate the plate/tube(s) on the magnet at room temperature for ≥ 30 sec.
    5.8
    Carefully remove and discard the ethanol.
    5.9
    Keeping the plate/tube(s) on the magnet, add 200 μL of 80% ethanol.
    5.10
    Incubate the plate/tube(s) on the magnet at room temperature for ≥ 30 sec.
    5.11
    Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
    5.12
    Dry the beads at room temperature for 3 – 5 min, or until all of the ethanol has evaporated. Caution: over-drying the beads may result in reduced yield.
    5.13
    Remove the plate/tube(s) from the magnet.
    5.14
    Thoroughly resuspend the beads in an appropriate volume of elution buffer (10 mM Tris-HCl, pH 8.0 – 8.5) or PCR-grade water. Always use PCR-grade water if proceeding to target capture.
    5.15
    Incubate the plate/tube(s) at room temperature for 2 min to elute DNA off the beads.
    5.16
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    5.17
    Transfer the clear supernatant to a new plate/tube(s) and proceed with Library Quantification (step 6). Store purified, amplified libraries at 2 °C to 8 °C for 1 – 2 weeks, or at -15 °C to -25 °C.

  6. Library Quantification
    Accurate quantification of amplifiable library molecules is critical for the efficient use of next-generation sequencing platforms. Overestimation of library concentration results in lower cluster density after bridge PCR. Underestimation of library concentration results in too many clusters on the flow cell, which can lead to poor cluster resolution. Both scenarios result in suboptimal sequencing capacity. Accurate library quantification is equally important when pooling indexed libraries for multiplexed sequencing to ensure equal representation of each library. Integrate KAPA Real-time Library Amplification Kit with the appropriate KAPA Library Quantification Kit to accurately quantify the number of PCR competent molecules. If libraries have been terminated between Fluorescent Standards 1 – 3, a single 1:1,000 dilution of each library will be required for library quantification using the KAPA Library Quantification Kits.

    Integrate KAPA Real-time Library Amplification Kit with the appropriate KAPA Library Quantification Kit to accurately quantify the number of PCRcompetent molecules. If libraries have been terminated between Fluorescent Standards 1 – 3, a single 1:1,000 dilution of each library will be required for library quantification using the KAPA Library Quantification Kits.

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