KAPA hgDNA Quantification and QC Kit

Quality matters.

KAPA hgDNA Quantification and QC Kits contain all the reagents needed for the qPCR-based quantification and quality assessment of human genomic DNA samples prior to NGS library construction.

Kits contain KAPA SYBR® FAST qPCR Master Mix, optimized for high-performance SYBR Green I-based qPCR, as well as a pre-diluted set of DNA standards and primer premixes targeting different portions of a highly conserved single-copy human locus. Absolute quantification is achieved with the primer pair defining the shortest fragment, whereas the additional primers are used to derive information about the amount of amplifiable template in the DNA sample. Quality scores (or Q-ratios) generated with the kit may be used to predict the outcome of library construction, or tailor workflows for samples of variable quality, particularly FFPE DNA.

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.

Download our KAPA hgDNA Quantification and QC Data Analysis Template.

For Research Use Only. Not for use in diagnostic procedures.

Product Highlights

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 is indicative of DNA quality, or the amount of amplifiable material in a DNA sample. Data on file.

Obtain concentration and quality information with a single assay

  • Allows for accurate quantification of dilute DNA samples
  • Quantification with an additional primer pair provides a Q-ratio that is indicative of sample quality
Conversion rates (% input DNA converted to adapter-ligated library) for libraries prepared for targeted re-sequencing on the Illumina platform. Libraries were prepared in duplicate from 80 – 100 ng FFPE DNA of variable quality (orange) or a commercial hgDNA preparation (grey), using the KAPA LTP Library Preparation Kit. FFPE DNA samples were assessed with the KAPA hgDNA Quantification and QC assay prior to Covaris shearing. Q-ratios were as indicated. The amount of fragmented DNA (average size ~180 bp) used for library construction was determined using the Qubit HS dsDNA assay (Life Technologies), whereas adapter-ligated libraries were quantified using the qPCR-based KAPA Library Quantification Kit. Results indicated that conversion rates for FFPE samples were significantly lower than for high-quality DNA. This limits the diversity of FFPE libraries, and necessitates more cycles of library amplification to generate sufficient material for target capture. This results in higher duplication rates and lower target coverage for FFPE libraries. Data on file.

Employ quality scores in the analysis of NGS library construction workflows

  • Q-ratios can provide valuable insights into the bottlenecks in NGS library construction workflows
  • For FFPE samples, library and sequence quality is primarily limited by inefficient conversion of input DNA to adapter-ligated library

Obtain actionable data for sample preparation

  • Q-ratios correlate with key sequencing metrics such as duplication rates and mean target coverage
  • FFPE samples with a Q score >0.4 yield libraries of acceptable quality when processed in standard sample preparations for target capture
Applications
  • FFPE samples
  • Free-circulating DNA from plasma or serum
  • Samples obtained by laser-capture microdissection of fresh, frozen or FFPE tissue
  • Forensic samples
  • Cells collected by flow cytometry
  • Any other limiting or precious clinical sample
Kit Specifications and Contents / Storage

Kits can be stored for up to 12 months at -20˚C.

Complete (Master Mix) kits include KAPA SYBR FAST qPCR Master Mix (2X), Primer Premix (41 bp, 129 bp and 305 bp, 10X) and a set of 5 DNA Standards. Primer Premixes and DNA Standards are also sold separately.

Components

Specifications

Spec
Description
Compatible Platform
All NGS platforms
Compatible Samples
Human genomic DNA
Sample sources
FFPE tissue Cells collected by laser-capture microdissection Flow-sorted cells Free-circulating DNA from plasma or serum Forensic samples Clinical samples
Standard curve concentration range
2.5 ng/µL–10 pg/µL or 3,080–12 copies
Sequencing Applications
Whole Genome Sequencing Whole Exome Sequencing Targeted Sequencing (custom panels) Amplicon Sequencing
FAQs
Why is quantitative PCR a better option than standard protocols for determining DNA input into library preparation?

Spectrophotometric and electrophoretic methods used routinely during NGS library construction (e.g. those employing a NanoDrop, PicoGreen, or Bioanalyzer) have significant limitations in terms of quantifying DNA input, which translate to poor prediction of library construction success. These limitations include:

  • low accuracy in the quantification of dilute samples;
  • inability to distinguish between total DNA, and DNA that can be utilized in amplification-based processes (e.g. library amplification, qPCR-based library quantification, cluster amplification) important for sequencing; and
  • sensitivity to contaminants, which can lead to significant over-or underestimation of DNA concentration.

What is a Q-ratio and how can it be used to assess DNA quality?

The same set of quantification 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. High quality hgDNA will have a Q129 bp/Q41 bp ratio around  1 or Q305 bp/Q41 bp ratio  around 1. Damaged DNA will have Q129 bp/Q41 bp ratio < 1 or Q305 bp/Q41 bp ratio << 1.

Q-ratios may be used:

  • to predict the outcome of library construction from FFPE and other clinical or limited samples of variable concentration and quality: Q-ratios have been shown to correlate with post-amplification yield, insert size, and library complexity;
  • for library construction process control and optimization: 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; and
  • to detect hgDNA contamination in free-circulating DNA samples.

What are the main sources of error and variability in the quantification and quality assessment of dilute hgDNA sample?

  • Inaccurate liquid handling: Take care to ensure the highest degree of accuracy during the dilution of hgDNA samples and reaction setup.
  • Reaction volumes: While the standard protocol for this assay is 20 µL reactions, the reaction volume can be scaled down to 10 µL. In this regard, accurate liquid handling is particularly important.
  • Sample quality: Absolute concentrations calculated for very dilute and/or damaged DNA samples Q129 bp/Q41 bp ratio less than around 0.2 may not be accurate or reproducible. Store hgDNA samples in a weak buffer (10 mM Tris-HCl, pH 8.0). Tween-20 should be included at a final concentration of 0.05% to improve pipetting accuracy. hgDNA dilutions should be made fresh and kept on ice while qPCRs are set up.
  • Sample concentration: Ideally samples should be diluted to fall within a range of 0.1 – 1.0 ng/µL. If more than 10 ng of DNA is added to a 10 µL reaction, problems with data collection and/or analysis may be experienced as a result of excessively high initial background fluorescence.
  • Incorrect product storage and/or excessive freezing and thawing: Observe all storage and handling guidelines in the Product Specifications section of the User Guide.
  • Contamination: Observe good laboratory practice at all times to avoid contamination of reagents, samples, consumables, pipettes and other equipment, and work areas with hgDNA or amplicons generated with this assay.
  • Consumables and equipment: Use high-quality PCR tubes/plates and pipette tips.

Do I need to perform replicates with my qPCR reactions?

qPCR is an extremely sensitive measurement technique that is vulnerable to variation arising from a number of sources. Even if the greatest attention is paid to liquid handling, inherent sources of variability such as instrument performing and sampling error lead to unavoidable scatter among replicate data points. Triplicate qPCRs are recommended and are generally sufficient for standard curve data points and for sample dilutions in the concentration range of Standard 1–3 (2,500 pg/µL–156 pg/µL).  However, for reliable assessment of samples in the concentration range of Standards 4–5 (39.1 pg/µL–9.77 pg/µL) you may wish to include additional qPCR replicates, according to the level of accuracy required.

Should I include a melt curve analysis with my qPCR cycling set-up?

Each of the three amplicons generated with this kit has a specific melting temperature and a characteristic melting profile. We recommend that a melt curve analysis be performed to confirm that specific product has been amplified in each reaction.

What are the storage recommendations for KAPA hgDNA Quantification and QC Kit?

We recommend that all reagents are stored protected from light at -20°C when not in use. Nevertheless, these reagents are stable in the dark at 4°C for at least one week, and may be stored in this state for short-term use, provided that they do not become contaminated with microbes and/or nucleases.  All components of KAPA hgDNA Quantification and QC Kit are stable through 30 freeze/thaw cycles.

Ordering

Complete (Master Mix) kits include KAPA SYBR FAST qPCR Master Mix (2X), Primer Premix (41 bp, 129 bp and 305 bp, 10X) and a set of 5 DNA Standards. Primer Premixes and DNA Standards are also sold separately.

Complete kits

Kit Code
Roche Cat. No
Description
Kit Size
How to buy
KK4960
07960590001
qPCR Master Mix (Universal)
300 x 20 µL reactions
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KK4961
07960603001
qPCR Master Mix (ABI Prism®)
300 x 20 µL reactions
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KK4962
07960611001
qPCR Master Mix (Bio-Rad®)
300 x 20 µL reactions
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KK4969
07960689001
qPCR Master Mix (ROX Low)
300 x 20 µL reactions
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KK4963
07960620001
qPCR Master Mix (optimized for Roche® LightCycler 480)
300 x 20 µL reactions
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Components

Kit Code
Roche Cat. No
Description
Kit Size
How to buy
KK4965
07960646001
10X 41 bp Primer Premix only
200 µL
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KK4966
07960654001
10X 129 bp Primer Premix only
200 µL
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KK4967
07960662001
10X 305 bp Primer Premix only
200 µL
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KK4964
07960638001
DNA Standards (1 - 5) only
5 x 300 µL
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