The Library Quantification Kit includes all the reagents needed for absolute quantification of NGS libraries via qPCR. It provides a reliable, qPCR-based quantification method for Illumina® and Ion TorrentTM sequencing libraries. All reagents provided in the kit undergo strict quality control to ensure maximum repeatability of experimental results across different batches.

Principle of Operation

        The kit utilizes qPCR reactions with target adapter sequences, platform-specific primers, and DNA standards to generate a standard curve based on the average Cq values of known-concentration DNA standards (left image). This standard curve is then used to convert the average Cq values of diluted libraries into concentrations, thereby determining the concentration of each library.

        This product is designed for absolute quantification of high-throughput sequencing library concentrations on 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 concentrations not lower than 0.006 pM, this product can be used for absolute quantification. Additionally, this product can also be used to detect library contamination levels in the experimental environment.

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

Differences between qPCR Library Quantification and 2100\Qubit Detection Results

        As shown in the figure: During the construction of NGS sequencing libraries, there may be cases of adapter dimers being ligated singly or some fragments not being ligated with adapters at all. This introduces the risk of errors during quantification before sequencing, leading to uneven sample mixing, reduced data volume, or biases in the data.

        Currently, due to limitations of detection platforms, Agilent Bioanalyzer 2100, Qubit (PicoGreen), or Nanodrop cannot distinguish whether sequencing adapters have been correctly added, and the results obtained represent the actual DNA concentration rather than the effective (with adapters) DNA concentration. The qPCR library quantification kit amplifies and detects effective DNA concentration using primers that perfectly match the sequencing adapters. By establishing a standard curve and accurately calculating the concentration of the products, uniform sequencing data can be achieved.