The product is a library quantification assay kit that contains all the reagents required for qPCR-based absolute quantification of NGS libraries. It provides a reliable quantitative solution for Illumina® and Ion TorrentTM platform sequencing libraries based on qPCR. All reagents provided in the kit undergo strict quality control to ensure maximum repeatability of experimental results between different batches.

Principle of Operation:

        Using qPCR with target adapter sequences, platform-specific qPCR primers, and DNA standards, the average Cq value of known concentration DNA standard is determined (see left image), generating a standard curve. This allows conversion of the average Cq value of the diluted library to concentration, thereby determining the concentration of each library.

        This product is designed for absolute quantification of high-throughput sequencing library concentration 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 no longer than 1 kb, and have a concentration of no less than 0.006 pM, this product can be used for absolute quantification. Additionally, this product can also be used to detect library contamination in the experimental environment.

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

        As shown in the figure, during NGS library construction, there may be cases of single-end adapter joining or some fragments without adapters, leading to errors in quantification before sequencing. This can result in uneven mixing of samples, low data yield, or bias in sequencing. Currently, due to limitations of the detection platform, Agilent Bioanalyzer 2100, Qubit (PicoGreen), or Nanodrop cannot distinguish whether sequencing adapters have been correctly added. The measured results represent the actual DNA concentration rather than the effective (containing adapters) DNA concentration. The qPCR library quantification assay, by amplifying and detecting with primers that perfectly match the sequencing adapters, determines the concentration of effective DNA through establishing a standard curve, thereby accurately calculating the concentration of the product and achieving uniform data.