in Application Notes, Articles, Sequencing

A PCR-based absolute quantification assay for sensitive and reliable quantification of NGS libraries prepared for Illumina® sequencing.

Next-generation sequencing (NGS) workflows are rapidly evolving, driven by users who require greater reliability, throughput and efficiency. Indeed, accurate quantification of PCR-competent sequencing templates is crucial for clonal amplification.

However, standard protocols for all major platforms currently rely on laborious, costly and unreliable methods for quantifying library molecules.

Using standard methods for quantifying libraries prepared for sequencing on the Illumina Genome Analyzer poses the following disadvantages:

  • These methods have a low sensitivity.
  • Electrophoresis and spectrophotometry measure total nucleic concentrations, while optimal cluster density depends on the appropriate concentration of PCR-amplifiable DNA molecules. For reliable optimization, expensive and time-consuming bPCR titrations are required.
  • Electrophoresis and spectrophotometry are not suited for high-throughput samples, requiring laborious and error-prone manual liquid handling.

 

Quantitative PCR

In principle, quantitative PCR (qPCR) is well-suited for NGS library quantification.1,2 It overcomes many of the difficulties inherent in the standard approaches:

  • Quantitative PCR quantifies only amplifiable library molecules.
  • It is accurate across an extremely large dynamic range.
  • It is suitable for high sample throughput and automated liquid handling.

However, for qPCR assays for NGS library quantification to be accurate and reproducible, there are a series of requirements:

  • A reliable supply of well-defined DNA standards, with minimal variation over time.  
  • Reagents capable of efficient amplification of long and complex templates.
  • Traditional qPCR reagents are optimized for short amplicons; longer targets, unbalanced GC-content, and problematic secondary structures may result in unreliable quantification of some library molecules.

 

qPCR-based NGS Library Quantification Kits

To address these requirements, Kapa Biosystems has developed a suite of qPCR-based NGS Library Quantification Kits. These KAPA library kits are comprised of highly reproducible sets of ready-to-use, serially diluted DNA standards; and next-generation qPCR reagents, which include a DNA polymerase specifically engineered for robust, SYBR Green I-based amplification of long and difficult templates.

KAPA library kits combine a quality-controlled set of DNA quantification standards with the robust performance of qPCR reagents to provide a rapid, sensitive and reliable method for quantifying PCR-amplifiable molecules in next-generation sequencing DNA libraries. They address a key pain point in NGS sample preparation workflows, namely the ability to achieve optimal utilisation of sequencing capacity in each run.

Researchers of The Broad Institute in Cambridge, USA described their findings of the implementation of the KAPA Library Quantification. They show how it improves on the workflow and reduces cluster density variability for sequencing on the Illumina® GA IIx. Would you like to know more about this study? You can download the application note here.  

For research use only. Not for use in diagnostics procedures.

1. Meyer, M., Briggs, A. W., Maricic, T., and Ho, B. (2007). From micrograms to picograms: quantitative PCR reduces the material demands of high-throughput sequencing. Nucleic Acids Research 36(1): e5.

2. Quail, M. A., Kozarewa, I., Smith, F., Scally, A., Stephens, P. J., Durbin, R., et al. (2008). A large genome center’s improvements to the Illumina sequencing system. Nature Methods, 5(12): 1005 – 1010.