The Library Quantification Kit includes all the reagents necessary for absolute quantification of NGS libraries required for qPCR amplification. It provides a reliable qPCR-based quantification method for Illumina® and Ion TorrentTM sequencing library preparation. All reagents provided in the kit have undergone strict quality control, ensuring the repeatability of experimental results across different batches to the greatest extent possible.

Working Principle of the Kit

        The kit utilizes qPCR reactions with target adapter sequences, platform-specific primers, and DNA standard samples. By generating a standard curve from the average Cq values of known concentration DNA standard samples (as shown in the left image), the average Cq values of diluted libraries can be converted into concentrations, thereby determining the concentration of each library.

        This product is designed for absolute quantification of high-throughput sequencing library concentrations for the Illumina® platform. Regardless of the construction method used, as long as the library ends contain Illumina® p5 and p7 chip binding sequences, are less than 1 kb in length, and have a concentration not lower than 0.006 pM, this kit can be used for absolute quantification. Additionally, this product can also be used to detect the degree of library contamination in the experimental environment.

        p5: 5'-AATGATACGGCGACCACCGA-3' p7: 5'-CAAGCAGAAGACGGCATACGA-3'.

Figure: Discrepancy between qPCR library quantification and results from 2100/Qubit detection.

        As shown in the figure: During NGS library construction, there may be cases of adapter dimers or fragments without adapters being present, which poses a risk of errors during pre-sequencing quantification, leading to uneven mixing of samples, reduced data volume, or bias. Currently, due to limitations of the detection platforms, Agilent Bioanalyzer 2100, Qubit (PicoGreen), or Nanodrop cannot distinguish whether sequencing adapters have been correctly added. The results obtained represent the actual DNA concentration rather than the effective (with adapters) DNA concentration. The qPCR library quantification assay, by amplifying and detecting with primers perfectly matching the sequencing adapters, determines the concentration of effective DNA through establishing a standard curve, enabling precise calculation of product concentration, and subsequently achieving uniform sequencing data.