Current standard protocols for commercial next generation sequencing platforms employ laborious, costly, and often unreliable methods for quantifying DNA libraries.
Accurate quantification of PCR-competent sequencing templates is crucial for reliable clonal amplification via either emulsion PCR (emPCR) or bridge PCR (bPCR) – underestimation usually results in non-clonality, while overestimation can lead to inefficiency via poor yields of clonally amplified templates.
Standard methods for quantifying NGS libraries have a number of important disadvantages. Electrophoresis and spectrophotometry measure total nucleic acid concentrations, whereas optimal cluster density or template-to-bead ratio depend on the appropriate concentration of PCR-amplifiable DNA molecules. These methods also have low sensitivity, consuming nanograms of precious samples, and are not suitable for high-throughput workflows. Quantitative PCR (qPCR) is inherently well-suited for next-generation sequencing library quantification:
qPCR library quantification results in streamlined workflows
KAPA Library Quantification Kits eliminate the need for time-consuming and expensive titrations and provide a conducive format for streamlining high-throughput workflows.
Reliable quantification results in consistent cluster density
Efficient amplification of a wide range of templates during qPCR
Traditional qPCR reagents are optimized for short amplification targets; longer targets, unbalanced GC-content, and problematic secondary structures may result in low amplification efficiency and unreliable quantification of some library molecules.
To address the demands of quantifying complex DNA libraries, Kapa Biosystems has engineered a DNA polymerase specifically for SYBR® Green-based qPCR, enabling efficient amplification of targets that present a challenge to wild-type enzymes. KAPA Library Quantification Kits contain this engineered polymerase to ensure robust amplification of longer fragments, across a broad range of GC-content, required for accurate library quantification.
Reliable DNA quantification standards with minimal variability from lot-to-lot
qPCR Library Quantification results in streamlined workflows
For life science research only.
Not for use in diagnostic procedures.
Data on file.