To obtain high-quality Illumina next-generation sequencing (NGS) data, loading the flow cell with an appropriate amount of library DNA is essential. Loading an insufficient amount of library DNA will result in low cluster density and reduced sequencing yield; an overabundance of library DNA may increase cluster density and result in poor quality data. Standard methods of NGS library quantification, such as those based on electrophoresis or spectrophotometry, have low sensitivity, are non-specific for adapter-bound DNA, and typically require a large amount of library sample for analysis. The Library Quantification Kit provides a highly-sensitive, qPCR-based quantification method that specifically targets adapter sequences used for Illumina’s NGS reactions. This kit allows specific measurement of the sequencing library concentration even in samples that contain DNA molecules not bound to adapters. This kit can also be used to confirm adapter ligation during library preparation.
Library quantification with this kit is accomplished by qPCR using TB Green for detection. Amplification products are detected by intercalation of TB Green in double-stranded DNA. Fluorescence detection in real time allows quantification of amplification products. This kit includes DNA standards of known concentration to allow absolute quantification of library DNA.