Digital PCR Applications: Next-Generation Sequencing Library Quantification

Advances in next-generation sequencing (NGS) technologies have accelerated the discovery of actionable genomic targets. Accurate quantification of the final library products is critical to maximizing both data quality and output. However, conventional quantitative PCR (qPCR) methods commonly used to assess NGS library concentrations do not evaluate the concentration of complete library fragments. Moreover, in some cases, the amount of final library product is limited, and the input requirement for consistent qPCR quantification can become a hindrance.

Next-generation sequencing (NGS) libraries can be quantified with minimal sample handling and without the need to generate a standard curve using digital PCR. Multiple studies have demonstrated that utilizing digital PCR (dPCR) as a quantification tool for NGS libraries before sequencing helps optimize sequencing run performance, data generation, and data quality [1,2]. By leveraging multiplexed dPCR, users can identify and quantify specific library fragments representing sequence-able molecules (Figure 1).

This method enables accurate and precise library quantification, a critical step in both the Ion Torrent™ and Illumina® workflows, allowing for maximizing sequencing yields downstream. To achieve this high degree of precision, a TaqMan® Assay, designed to span both the forward and reverse adapters specific to each library, is available. Ultimately, using digital PCR to quantify NGS libraries can decrease overall sequencing costs by helping ensure an accurate quantification upfront, minimizing the need to re-run or repeat sequencing of samples.

Figure 1. dPCR assay for NGS library quantification
(A) The duplex assay amplifies only libraries that have P5 and P7 adapters. (B) using dPCR, absolute quantification of sequenceable library fragments is possible by counting the total number of double-positive microchambers.

1. Robin JD et al. (2016) Comparison of DNA quantification methods for next generation sequencing. Sci Rep 6(1):24067. doi: 10.1038/srep24067 

2. White RA et al. (2009) Digital PCR provides sensitive and absolute calibration for high throughput sequencing. BMC Genomics 10(1):116. doi: 10.1186/1471-2164-10-116

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