In this protocol

适用说明: 本说明书适用于KAPA Human Genomic DNA Quantification and QC Kits (07960590001, 07960603001, 07960611001, 07960620001 与 07960689001), KAPA Human Genomic DNA Quantification and QC DNA Standards (07960638001), 及 KAPA Human Genomic DNA Quantification and QC Primer Premixes (07960646001, 07960654001与07960662001).
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Product Description

Both the quantity and quality of input DNA have a profound impact on the outcome of library construction for next- generation sequencing (NGS). A number of innovations have reduced the amount of input DNA required for successful library construction, enabling routine sequencing of human DNA from a wide variety of sample types. Such high-throughput NGS pipelines require streamlined protocols that reduce input requirements, lower costs, improve success rates, and minimize turn- around times. A reliable method for quantifying and qualifying input DNA prior to library construction offers several benefits for any library construction workflow:
● Standardization or normalization of input DNA results in more consistent library construction outcomes (e.g., yield, library quality) with a fixed-cycle library amplification protocol, and reduces the need for sample-specific protocol adjustments.
● Quality metrics can be used to direct samples into appropriate, optimizedshearingandlibraryconstruction pipelines, to ensure more consistent insert sizes and data quality. Identifying the most appropriate workflow for each sample at the outset minimizes reworking of samples, and reduces average turn-around times.

Quantification methods that rely on spectrophotometry or electrophoresis (e.g., those employing a NanoDrop, Qubit or Bioanalyzer) have significant limitations for assessing input DNA, and provide poor predictions of library construction success.

Such limitations include:
● poor accuracy in the quantification of very dilute samples;
● the inability to discriminate between damaged DNA and template material suitable for PCR-based processes such as library amplification, qPCR-based library quantification, cluster amplification and sequencing; and
● sensitivity to contaminants, which can lead to significant over- or underestimation of DNA concentrations.

To address these needs, we have developed a qPCR- based kit for the reliable quantification and quality assessment of human genomic DNA (hgDNA) samples prior to NGS library construction. The kit employs the engineered KAPA SYBR® FAST DNA Polymerase, and includes three primer premixes and a set of quality- controlled, pre-diluted DNA quantification standards. The primer premixes are designed to amplify targets of 41 bp, 129 bp, and 305 bp within a conserved single- copy locus in the human genome.

Absolute quantification is achieved using the 41 bp assay, while the longer amplicons are used to assess DNA quality (Figure 1). Since DNA damage has a greater impact on the amplification of longer targets, the relative quality of a DNA sample can be inferred by normalizing the concentration obtained using the 129 bp or 305 bp assay against the concentration obtained with the 41 bp assay. This normalization generates a “Q-ratio” with a value between 0 and 1, which can be used as a relative measure of hgDNA quality prior to NGS library construction.


Figure 1. Principle of the hgDNA Quantification and QC assay. A single set of DNA Standards is used to generate up to three standard curves, using three different primer pairs that amplify targets of 41 bp, 129 bp, or 305 bp within a conserved, single-copy human locus. The 41 bp assay is used for absolute quantification of DNA samples. For an assessment of DNA quality, standard curves are generated and samples assayed with the 129 bp and/or 305 bp primer premix(es). Since poor DNA quality has a greater impact on the amplification of longer targets, the relative quality of a DNA sample can be inferred by normalizing the concentration obtained using the 129 bp or 305 bp assay against the concentration obtained from the 41 bp assay. This normalization generates a “Q-ratio” (with a value between 0 and 1) that can be used as a relative measure of DNA quality.

Product Applications

The KAPA Human Genomic DNA Quantification and QC Kit is designed for the reliable quantification and quality assessment of hgDNA samples prior to NGS library construction. These include:
●formalin-fixed paraffin-embedded (FFPE) tissue samples, which are notorious for variable DNA quality as a result of degradation, cross-linking, and/or other lesions that render DNA recalcitrant to amplification
● samples obtained by laser-capture microdissection of fresh, frozen, or FFPE tissue
● DNA extracted from cells collected by flow cytometry
● free circulating DNA from plasma or serum
● any other low concentration or precious sample.

Q-ratios may be used:
● to predict the outcome of library construction from FFPE and other difficult or limited samples of variable concentration and quality. Q-ratios have been shown to correlate with post-amplification yield, insert size, and library complexity
● forlibraryconstructionprocesscontrolandoptimization. Q-ratios can be used to assess DNA fragmentation prior to library construction; to make stop/go decisions for library construction based on sample quality; or for sample “triage”, to direct samples into the appropriate workflows
● to retrospectively troubleshoot failed samples, or
samples that produce substandard sequencing results
● to detect hgDNA contamination in free circulating DNA samples.

The method is easy to automate and can be applied to any process or workflow that requires accurate quantification of dilute DNA samples, or samples that may contain a high proportion of DNA that is recalcitrant to PCR amplification.

KAPA SYBR® FAST is an antibody-mediated hotstart DNA polymerase formulation. KAPA Human Genomic DNA Quantification and QC Kits are therefore suitable for use with automated liquid handling systems for high- throughput sample quantification.

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).
    1.2
    Ensure that all components are completely thawed, thoroughly mixed, and then briefly centrifuged.
    1.3
    Three 1 mL aliquots of KAPA SYBR® FAST qPCR Master Mix (2X) are supplied. When the kit is first used, add 200 μL of 41 bp Primer Premix (10X) to one tube of KAPA SYBR FAST qPCR Master Mix (2X), and label the tube accordingly. Similarly, add 200 μL of 129 bp Primer Premix (10X) to the second tube, and add 200 μL of 305 bp Primer Premix (10X) to the third tube, labelling the tubes appropriately. Mix thoroughly using a vortex mixer, and then briefly centrifuge the tubes.
    1.4
    Record the date on which the primers were added. KAPA SYBR FAST qPCR Master Mixes with primers are stable through 30 freeze/thaw cycles, and should be stored in the dark at -15°C to -25°C when not in use. Alternatively, the mixes may be stored in the dark at 2°C to 8°C for up to one week, provided that they do not become contaminated with microbes and/or nucleases.

  2. Sample Preparation
    2.1
    hgDNA samples must be diluted to fall within the dynamic range of the assay. Ideally, aim for a final DNA concentration of 0.1 – 1.0 ng/μL. Fresh dilutions should be made in Dilution Buffer as described in Reagent Preparation (step 1.1) and kept on ice during reaction setup. Diluted DNA stored at room temperature, or for prolonged periods at 2°C to 8°C, is prone to degradation and may not yield consistent results upon re-quantification. DNA diluted in water may be unstable, even when stored at room temperature for short periods of time.

  3. Reaction Setup
    3.1
    Prepare a separate master mix for each primer set to be used. Based on the reaction setup outlined below, determine the volume of master mix that will be required for triplicates of each of the following reactions: five DNA Standards, one no-template control (NTC), and each of the hgDNA samples to be assayed. Remember to provide for an excess of at least 10%.

    For Universal qPCR Master Mix:

    *Please refer to the KAPA SYBR FAST Technical Data Sheet included with your kit to select the appropriate ROX for your qPCR instrument.

    For ABI Prism, Bio-Rad iCycler, or LightCycler® 480 qPCR Master Mix:


    3.2
    Mix and briefly centrifuge each master mix.
    3.3
    Label the appropriate number of PCR tubes or decide on the appropriate plate layout.
    3.4
    Dispense 16 µL of the appropriate master mix (prepared in step 3.1) into each PCR well/tube.
    3.5
    Add 4 µL of the appropriate template (DNA Standard, diluted DNA sample from Sample Preparation (step 2) or PCR-grade water for NTCs) into each PCR well/tube. Use a fresh tip each time, and flush the tip by carefully pipetting up and down several times. Dispense the DNA Standards starting from the most dilute (Standard 5) and proceeding to the most concentrated (Standard 1).
    3.6
    Cap tubes or seal the PCR plate, and transfer to the qPCR instrument.
    3.7
    Select the Absolute Quantification option in the instrument software, and select/adjust parameters (e.g., reporters, reference dyes, gain settings, etc.) as required. Refer to the instrument manual for further information.
    3.8
    Refer to Data Analysis (step 4.1) for annotation of DNA Standards, if required at this stage.
    3.9
    Perform qPCR using the following cycling parameters:
      
    *See Important Parameters (p. 4) for more details.


  4. Data Analysis
    4.1
    Annotate the DNA Standards as outlined in the table below. The DNA Standards comprise a 4-fold dilution series of a 610 bp dsDNA template. Note that the specified values have already been converted to the concentration of hgDNA that is equivalent to the copy number corresponding to each DNA Standard.


    4.2
    Review the background-subtracted (normalized) amplification curves and the Cq scores for replicate data points, and exclude obvious outliers. Replicate data points for Standards 1 – 3, and samples in that concentration range, should differ by ≤0.2 cycles, while replicates for Standards 4 – 5, and samples in that concentration range, may vary by up to ~0.5 cycles.

    If the data set contains many outliers, the results of the assay are unlikely to be reliable, and the assay should be repeated with particular focus on improving pipetting accuracy.

    Exclude all samples that fall outside the range of the standard curve, i.e., that return an average Cq score lower than that of Standard 1, or higher than that of Standard 5. Where possible, such samples may be re-tested after preparing new dilutions at appropriate concentrations.
    4.3
    To generate the 41 bp standard curve and calculate sample concentrations, select only those Standards and samples that were amplified using the 41 bp Primer Premix, and deselect all data points generated with the 129 bp or 305 bp Primer Premixes.
    4.4
    Allow the software to generate the 41 bp standard curve, and to calculate the concentrations of the hgDNA samples. The standard curve and sample concentrations may also be generated manually, using a spreadsheet. For assistance in this regard, please contact sequencing.roche.com/support. 
    4.5
    Review the metrics for the 41 bp standard curve. The standard curve should be used only if the following conditions are met:
    ● R2 ≥0.99.
    ● The calculated reaction efficiency is 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). If the 41 bp standard curve does not meet these criteria, concentrations calculated for the hgDNA samples will not be reliable, and the assay should be repeated.

    A consistent reaction efficiency (within the range of 90 – 110%) should be achieved when the assay is performed on the same qPCR cycler, using the same plasticware. If the assay returns a reaction efficiency that is within the acceptable range, but is much higher or lower than what is routinely achieved, this may be an indication of unexpected experimental variation, and should be considered before accepting results.
    4.6
    Depending on the extent of the data analysis provided by your qPCR software, you may wish to export the qPCR data to a spreadsheet to carry out further calculations, as follows:
    ● Calculate the average concentration (from triplicate qPCRs) for each hgDNA sample dilution.
    ● Take account of any dilution performed in Sample Preparation (step 2), to calculate the concentration of each undiluted sample—see Working Example (step 5).

    4.7
    Repeat steps 4.3 – 4.6 to generate the standard curves and calculate the sample concentrations for the 129 bp and/or 305 bp assays.
    4.8
    To perform a quality analysis of hgDNA samples, calculate Q-ratios as follows:
    Q129/Q41 ratio: For each sample, divide the concentration calculated using the 129 bp assay by the concentration calculated using the 41 bp assay.
    Q305/Q41 ratio: For each sample, divide the concentration calculated using the 305 bp assay by the concentration calculated using the 41 bp assay.

  5. Working Example
    Two hgDNA samples were assessed using the KAPA hgDNA Quantification and QC Kit. One sample consisted of intact hgDNA, whereas the other contained hgDNA sheared to an average size of ~300 bp, representing damaged and/ or fragmented DNA. The concentrations of both samples were determined using a NanoDrop spectrophotometer, prior to dilution and qPCR analysis (Table 3, row 1).

    qPCR was performed in triplicate as described in Reaction Setup (step 3) using the 41 bp, 129 bp and 305 bp Primer Premixes. The following calculations were performed for the 41 bp, 129 bp, and 305 bp assays:
    ● Using the qPCR instrument software, the concentration of each diluted hgDNA sample was calculated from the appropriate standard curve as shown in Data Analysis (step 4.5). Values for each replicate qPCR are provided in Table 3, row 5).
    ● The average concentration of each diluted sample was calculated using a spreadsheet (see Data Analysis (step 4.6), and Table 3, row 6).
    ● Next, using the same spreadsheet, the average concentration of each undiluted sample was calculated (see Data Analysis (step 4.6), and Table 3, row 7).
    ● Finally, Q129/Q41 and Q305/Q41 ratios were calculated for each sample (see Data Analysis (step 4.8), and Table 3, rows 8 – 9).

    The Q-ratios illustrate that high-quality, unfragmented hgDNA (represented by the intact DNA sample) yields Q129/Q41 and Q305/Q41 ratios approaching ~1.0, whereas damaged/ fragmented hgDNA (represented by the sheared DNA sample) return lower Q ratio scores, with values <1. For such samples, it is expected that Q129/Q41 ≥ Q305/Q41.
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