In this protocol

适用说明:本说明书适用于KAPA Library Quantification Kits for Ion Torrent platforms (07960158001, 07960212001,07960263001, 07960344001 与 07960301001), KAPA Library Quantification Primer and PCR Mix Kits for Ion Torrent platforms (07960450001, 07960492001, 07960549001, 07960735001与07960581001), KAPA Library Quantification Standards and Primer Kit for Ion Torrent platforms (07960107001), KAPA Library Quantification Standards Kit for Ion Torrent platforms (07960395001), 和 KAPA Library Quantification Primer Kit for Ion Torrent platforms (07960115001).
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Product Description

Accurate quantification of amplifiable library molecules is critical for the efficient use of the Ion Torrent next generation sequencing (NGS) platform—overestimation results in too few DNA-bearing beads, while underestimation leads to multiple template molecules per bead during emPCR. Accurate library quantification is equally important when pooling indexed libraries for multiplexed sequencing to ensure equal representation of each library.

qPCR is inherently well-suited to NGS library quantification, as it overcomes many of the difficulties posed by alternative approaches, for example it:
● quantifies only the PCR-amplifiable library molecules that are relevant to optimizing emPCR,
● is exceptionally sensitive and accurate across an extremely broad dynamic range, and
● is amenable to high sample throughput and automated liquid handling. The extreme sensitivity of qPCR enables accurate quantification of very dilute libraries. This minimizes the need for PCR amplification of libraries and the associated problematic biases.

KAPA Library Quantification Kits are comprised of DNA Standards (six 10-fold dilutions) and Primer Premix (10X), paired with KAPA SYBR® FAST qPCR Kits to accurately quantify the number of amplifiable molecules in an NGS library. The 153 bp KAPA Ion Torrent DNA Standard consists of a linear DNA fragment flanked by qPCR primer binding sites. Quantification is achieved by inference from a standard curve generated using the six DNA Standards.

KAPA Library Quantification Kits are vigorously tested to ensure minimal lot-to-lot variation. The kit contains the novel KAPA SYBR FAST DNA Polymerase, engineered through a process of directed evolution for high- performance SYBR Green I-based qPCR. The ability of the engineered polymerase to amplify diverse DNA fragments with similar efficiency enables the use of a universal standard for the reliable quantification of libraries with an average fragment length of up to 1kb, irrespective of library type or GC content. KAPA Ion Torrent Library Quantification Kits are suited for the quantification of libraries constructed with Ion Torrent adapters containing the following qPCR primer sequences:
Primer IT A: 5'-CCA TCT CAT CCC TGC GTG TC - 3'
Primer IT trP1: 5'-CCT CTC TAT GGG CAG TCG GTG AT-3'

Product Applications

KAPA Library Quantification Kits are suitable for any application requiring sensitive, accurate, reproducible, and/ or high-throughput NGS library quantification, including:
● high-throughput library construction and quantification pipelines, especially where automation is desirable;
● pooling of indexed libraries for multiplexed sequencing, to ensure equal representation of pooled samples in sequencing data; and
● optimizing input DNA concentrations for reliable emPCR.

Process Workflow

Detailed Protocol

  1. Reagent Preparation
    1.1
    Prepare an appropriate volume of DNA Dilution Buffer [10 mM Tris-HCl, pH 8.0 – 8.5 (25 °C) + 0.05% Tween® 20 (optional)]. This buffer can be stored at room temperature or 4 °C, and re-used. Always equilibrate the buffer to room temperature before use.
    1.2
    Ensure that all components of the KAPA Library Quantification Kit are completely thawed and thoroughly mixed.
    1.3
    If the kit is used for the first time, add the Primer Premix (10X) (1 mL) to the bottle of KAPA SYBR® FAST qPCR Master Mix (2X) (5 mL). Mix thoroughly using a vortex mixer.

     If you are using the Universal qPCR Master Mix Kit and will only use ROX High or ROX Low, the appropriate ROX solution (50X) (0.2 mL) may be added to the qPCR Master Mix with primers when the kit is first opened. The volume of this mixture used per reaction should be adjusted accordingly (12.4 µL per 20 µL reaction or 6.2 µL per 10 µL reaction).
    1.4
    Record the lot numbers of all reagents, as well as the date on which the primers (and ROX) were added to the qPCR Master Mix. KAPA SYBR FAST qPCR Master Mixes with primers (and ROX) are stable through 30 freeze-thaw cycles, and should be stored protected from light at -15°C to -25°C when not in use. Mixes may be stored in the dark at 2°C to 8°C for up to one week, provided that they are not contaminated with microbes and/or nucleases during preparation or subsequent use in reaction setup.

    Table 1. Recommended ROX concentrations for use with KAPA SYBR FAST Universal qPCR Master Mix


  2. Sample Preparation
    2.1
    Prepare an initial 1:500 dilution of the purified library (using DNA dilution buffer).
    2.2
    The 1:500 dilution may be prepared as follows:

    2.3
    Mix thoroughly by vortexing for 10 sec.
    2.4
    Prepare any additional library dilutions. Note: at least one additional 2-fold dilution of each library is recommended to ensure at least one dilution falls within the dynamic range of the standards.
    2.5
    Prepare the internal control dilutions (if applicable).

  3. Reaction Setup and Cycling
    3.1
    Determine the total number of reactions that will be performed for the appropriate number of replicates of each of the following reactions:
    ● six DNA Standards
    ● each dilution of every library to be assayed
    ● each dilution of any internal controls
    ● no-template controls (NTCs)
    3.2
    Prepare the required volume of master mix using the reaction setup recommended below.

    Reaction setup: 20 µL reactions

    1If ROX was added to the qPCR Master Mix and primers, use 12.4 µL of the qPCR Master Mix with Primer Premix and ROX per reaction.



    Reaction setup: 10 µL reactions

    1If ROX was added to the qPCR Master Mix and primers, use 6.2 µL of the qPCR Master Mix with Primer Premix and ROX per reaction.
    2The recommended reaction setup results in a total reaction volume of 10.2 µL if ROX is added during reaction setup. This does not impact performance.



    3.3
    Mix and briefly centrifuge the reagent master mix.
    3.4
    Dispense the appropriate volume of the master mix into each PCR tube or well.
    3.5
    Add 4 µL of PCR-grade water to all NTC tubes/wells.
    3.6
    Dispense 4 µL of each DNA Standard into the appropriate tubes/wells, working from the most dilute (Standard 6) to the most concentrated (Standard 1).
    3.7
    Dispense 4 µL of each dilution of libraries and internal controls to be assayed.
    3.8
    Cap tubes or seal the PCR plate, and transfer to the qPCR instrument.
    3.9
    Perform qPCR with the following cycling protocol, selecting the Absolute Quantification option in the instrument software. Adjust run parameters (e.g., reporters, reference dyes, gain settings, etc.) as required.


    *Optional; see Important Parameters for more details.

  4. Data Analysis
    In general, when standard libraries are quantified by qPCR, optimal DNA input for emPCR is
    0.2 – 1 copies/bead, with the optimal input concentration varying from lab to lab according to sample type, library construction, etc. If the emPCR has been previously optimized using a different library quantification method and now qPCR is to be used, then it may be necessary to determine the optimal loading concentration (copies/bead) using qPCR-derived library concentrations. This can be accomplished empirically either via qPCR library quantification followed by emPCR titrations, or by using qPCR to retrospectively quantify a number of representative libraries that have already been used successfully in emPCR. For more information, please contact Technical Support at sequencing.roche.com/support.
    4.1
    Annotate the DNA Standards as outlined below. Note that the specified values correspond to the concentrations of the DNA Standards, and not the final DNA concentration in each reaction. It is not necessary to convert these to the actual concentrations in the reaction, as long as the same volume of template (DNA Standard, diluted library or internal control) is used in all reactions.


    4.2
    Review the background-subtracted (normalized) amplification curves and the Cq scores for replicate data points (DNA Standards, libraries and controls), and exclude obvious outliers. Replicate data points should differ by ≤0.2 cycles. If the data set contains many outliers, results are unlikely to be reliable. Repeat the assay with particular focus on improving pipetting accuracy.
    4.3
    Exclude all library dilutions that fall outside the dynamic range of the assay, i.e., that return an average Cq score lower than that of Standard 1 or higher than that of Standard 6. If all the dilutions of a library fall outside the standard curve, re-quantify a more appropriate dilution of the library.
    4.4
    Use the instrument software to generate the standard curve. The standard curve may also be generated manually using the KAPA Library Quantification Data Analysis Template for Ion Torrent.
    4.5
    Review the standard curve to ensure that the following criteria are met:
    ● The average ΔCq value between DNA Standards is in the range of 3.1 – 3.6.
    ● The calculated reaction efficiency is in the range of 90 – 110% (i.e., the PCR product has increased 1.8- to 2.2-fold per cycle, and the slope of the standard curve is between -3.1 and -3.6).
    ● R2 ≥0.99.

    If the standard curve does not meet these criteria, calculated library concentrations will not be reliable, and the assay must be repeated.
    4.6
    Most qPCR software will calculate the concentration of the library and control dilutions using absolute quantification against the standard curve. However, we recommend exporting qPCR data to the KAPA Library Quantification Data Analysis Template for Ion Torrent to perform the following calculations to determine the undiluted library concentration:
    ● Use the standard curve to convert the average Cq score for each dilution of every library and internal control that was assayed to average concentration (in pM).
    ● Calculate the average size-adjusted concentration (in pM) for each dilution of every library and control that was assayed, by multiplying the calculated average concentration with the following factor:


    ● Multiply the average size-adjusted concentration calculated for each dilution of every library or control that was assayed with the appropriate dilution factor to calculate final concentration for the undiluted library or control from each of the dilutions assayed.
    4.7
    Review the final calculated concentrations and determine the working concentration for each sample to be used for downstream emPCR.

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