KAPA hgDNA Quantification and QC Kit

• 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
• Forensic samples
• Any other low-concentration or precious clinical sample

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.

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 Q_{129 bp}/Q_{41 bp} ratio around  1 or Q_{305 bp}/Q_{41 bp} ratio  around 1. Damaged DNA will have Q_{129 bp}/Q_{41 bp} ratio < 1 or Q_{305 bp}/Q_{41 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.

• 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 Q_{129 bp}/Q_{41 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.

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.

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.

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.