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 clinically relevant 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, optimized shearing and library construction 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 qPCRbased 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 qualitycontrolled, pre-diluted DNA quantification standards. The primer premixes are designed to amplify targets of 41 bp, 129 bp, and 305 bp within a conserved singlecopy locus in the human genome.
Absolute quantification is achieved using the 41 bp assay, while the longer amplicons are used to assess DNA quality. 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.