Abstract
Accurate quantification of NGS libraries is critical for a successful sequencing run. Currently used methods of quantification are time-consuming, costly, and can be highly variable. We have developed NuQuant, a novel method to accurately quantify NGS libraries, that can be performed with a simple fluorescent measurement. NuQuant is compatible with the common red fluorescence excitation/emission filter set (650/670 nm), making it compatible with a wide range of bench top fluorometers and fluorescent plate readers. We have developed a custom library quantification application for the Qubit fluorometer that directly provides the molar concentration of a library. Utilizing this application, we have demonstrated that NuQuant has excellent reproducibility across users from multiple sites. We now demonstrate the compatibility of NuQuant with standard fluorescence plate readers, enabling quantification of libraries in a high-throughput fashion. We have tested NuQuant on a variety of commonly used plate readers such as the Tecan Infinite 200 Pro and the Promega GloMax. Libraries in a 96-well format can be measured in a matter of minutes, without the need for sample dilution. Molar concentration of libraries was easily determined by utilizing a standard curve. We tested libraries with various input from 10ng to 500ng and insert size from 200bp to 500bp, and found good agreement between NuQuant values and a qPCR based quantification method. Most importantly, we observe good correlation between NuQuant library concentration and total number of sequenced reads. In conclusion, scientists with access to commonly used fluorescent plate readers can now use NuQuant to achieve rapid and cost-effective quantification of NGS libraries, generating highly uniform sequence reads in multiplex runs.