Performance

The sensitivity and dynamic range of the GeneRead Library Quant System were compared against Agilents High Sensitivity DNA Kit, using two NGS libraries of differing concentrations. The first library was quantifiable by both systems, but the low concentration of the second library was below the detection limit of Agilents 2100 BioAnalyzer. By contrast, the GeneRead Library Quant Array was able to quantify both libraries (see figure QIAseq Library Quant System enables quantification of libraries with concentrations below the detection limit of conventional methods).

Principle

Accurate quantification of library molecules is essential for the efficient use of NGS platforms; however, current spectrophotometric methods only measure total nucleic acid concentrations. The GeneRead Library Quant System, by contrast, uses real-time PCR to quantify only DNA molecules with adaptors at both ends, which are the only amplifiable molecules during emulsion PCR (Ion Torrent platform) or bridge PCR (Illumina platform), and therefore provides highly accurate quantification of amplifiable library molecules. The high sensitivity of real-time PCR allows quantification of libraries at very low concentrations, even below the detection threshold of conventional spectrophotometric methods.

The GeneRead Library Quant System is available in both a tube and an array format, and is optimized with QIAseq qPCR SYBR^® Green Mastermixes to provide superior sensitivity and a wide dynamic range. Both formats provide a DNA standard specific to the Illumina or Ion Torrent platform. The array format provides this standard in five predispensed, sequential 10-fold dilutions mixed with a PCR primer assay in triplicate, ensuring that your sample library will fall within the detection range of the array (see figure Principle of QIAseq Library Quant System).

Procedure

The array format workflow (see figure QIAseq Library Quant Array workflow) begins with two tenfold dilutions of the sample library, to ensure that its concentration falls within the range of the serially diluted standards. The samples are mixed with QIAseq qPCR SYBR Green Mastermix and aliquoted in technical triplicate across the array plate. PCR is performed, and raw C_T values are exported to the provided data analysis Excel file for automatic calculation of library concentration (Illumina platform) or template dilution factor (Ion Torrent/Proton platform).

The tube format workflow is similar to the array format workflow, with a few small differences. The procedure begins with preparation of five sequential tenfold dilutions of DNA Standard and two tenfold dilutions of the sample library. This ensures that the concentration of the library will fall within the range of the standard dilutions. Next, the diluted DNA standards and sample libraries are mixed with the provided PCR assay and the appropriate QIAseq qPCR SYBR Green Mastermix. PCR is performed, and C_T values are exported to the provided data analysis Excel file for automatic calculation of library concentration (Illumina platform) or template dilution factor (Ion Torrent/Proton platform).

Applications

The GeneRead Library Quant Array and GeneRead Library Quant Kit provide rapid, accurate measurement of sample library concentration prior to sequencing with the Illumina or Ion Torrent/Proton NGS platforms.