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.