Accurate quantification of the number of amplifiable library molecules loading into a flow cell is one of the most critical steps in the next-generation sequencing (NGS) workflow in obtaining high-quality read data with NGS technologies. Loading 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, by electrophoresis or spectrophotometry, have low sensitivity, are non-specific for adapter-bound DNA and typically require a large amount of library sample for analysis. With its greater sensitivity and broad dynamic range, qPCR is therefore seen as the gold standard for NGS library quantification, as it accurately measures the number of molecules that can serve as templates during library and cluster amplification, even with very dilute libraries.
Bioline has developed the JetSeq™ Library Quantification Kit, an optimized, robust SYBR® Green based qPCR kit that provides accurate quantification of Illumina based NGS libraries. The JetSeq Library Quantification Kit contains pre-diluted standards to minimize pipetting errors, a pre-qualified P5 and P7 Illumina adaptor sequence primer mix to ensure reproducible and precise qPCR results and an optimized buffer for dilution of NGS library samples.
Accurate - qPCR-based assay for quantification of only adapter-ligated library molecules, thereby enabling optimal flow cell loading for maximum data yield and quality
Sensitive - reliable quantification of even low-yield libraries, ideal for both PCR and PCR-free library preparation methods
Fast - delivers accurate assay results in as little as 90 minutes, thereby reducing time to results
Convenient - contains a series of six pre-diluted DNA standards for rapid, simple standard curve development
Economical - contains sufficient reagents and standards to quantify eighteen individual libraries on separate plates
qPCR amplification of each of the six supplied DNA standards (blue) and a tenfold serial dilution of the dilued Illumina NGS library (red) were carried out with the supplied primer sets in triplicate A melt curve was performed to verify amplification of a single specific product for both standards and library even at the lowest concentrations From the resulting standard curve a size adjusted library concentration was then determined. The amplification plots show a limit of detection of 0.0001 pM (100 aM) Three different lots of the JetSeq Library Quantification Kit were compared by generating an amplification plot using the standards from each kit on the Mic qPCR Cycler. The results illustrate that the JetSeq Library Quantification Kit delivers exceptional accuracy in library quantification
Quantification of individual libraries or library pools prior to cluster amplification
Quantification of libraries prior to pooling for multiplexed sequencing
Quality control, optimization and troubleshooting of library construction processes or workflows