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:
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:
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.