Quantifying Next Generation Sequencing libraries using digital PCR on the Quantstudio3D platform

One of the challenges of Next Generation Sequencing (NGS) technologies is the accurate quantification of sequencing libraries prior to massively-parallel sequencing. Such quantifications are generally based on total DNA quantifications and molecular weight determinations. However, with the increasing use of new and novel library preparation methods and lower amounts of DNA/RNA used to create such libraries, better methods are needed to accurately quantify library molecules that contain all sequences necessary to function in the sequencers. Several methods are currently available, but probably the most accurate method to enumerate PCR-amplifiable molecules is digital PCR. We have developed primer and probe combinations to use the ABI QuantStudio3D digital PCR instrument to accurately quantify several types of NGS libraries. Retrospectively, the results of digital PCR correlate well with sequencing results and using these methods, prospectively, to quantify libraries should save time, money, and improve sequencing yields. Several strategies will be discussed to deal with the extremely low concentrations of library molecules used in the digital PCR reactions, and data from using an internal reference-based method will be described.