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For Research Use Only. Not for use in diagnostic procedures.
Overview
KAPA Library Quantification Kits contain all the reagents needed for the accurate, reliable and reproducible qPCR-based quantification of next-generation sequencing (NGS) libraries prepared for sequencing on Illumina and IonTorrent platforms. Kits include KAPA SYBR FAST qPCR Master Mix (formulated with different passive reference dyes for different qPCR instruments), a platform-specific library quantification primer premix, and a pre-diluted set of DNA standards.
Benefits of KAPA Library Quantification Kits
Reliable and sensitive quantification of all sequencing-competent library molecules
Accurate and reproducible quantitation across a wide range of library types, concentrations, fragment length distributions and GC content
Accurate, equimolar pooling for multiplexed sequencing
Flexibility to support manual and automated high-throughput pipelines as well as PCR-free workflows
Resources
What makes KAPA Library Quantification Kits highly efficient for library quantification?
KAPA Library Quantification Kits contain KAPA SYBR FAST DNA Polymerase, which was engineered through our directed evolution technology to amplify diverse DNA fragments with similar efficiency. This is important for the amplification of heterogeneous populations such as NGS libraries. Library quantification kits containing wild-type DNA polymerases only count “easy” library molecules.*
View data showing efficient amplification and quantification of diverse libraries by KAPA SYBR FAST DNA Polymerase compared to other options.
Figure 1. KAPA SYBR FAST exhibits high-efficiency amplification and library quantification. Amplification efficiencies achieved with KAPA SYBR FAST for ten diverse amplicons, plotted against GC content. All ten amplicons were amplified with similar efficiency (within the optimal range of 95% –105%). This confirms that KAPA SYBR FAST is the ideal enzyme for the amplification of heterogenous populations of DNA fragments, such as NGS libraries.
How do KAPA Library Quantification Kits Work?
Generation of standard curve and quantification of library concentration
Six pre-diluted DNA Standards and appropriately diluted NGS libraries are amplified using platform-specific qPCR primers that target adapter sequences. The average Cq value for each DNA Standard is plotted against its known concentration to generate a standard curve. The standard curve is used to convert the average Cq values for diluted libraries to concentration, from which the working concentration of each library is calculated.
Product highlights
The complete library solution
All reagents needed for absolute, qPCR-based quantification of individual NGS libraries or indexed library pools
Standard curves to support all library construction workflows, including PCR-free methods
Quantification of only/all sequencing-competent library fragments
Figure 3. DNA fragment size distributions for an NGS library before (grey fill) and after qPCR amplification with KAPA SYBR FAST and two competitor kits containing wild-type Taq polymerase. Reactions were performed with the following cycling protocol: 95°C for 10 min, followed by 40 cycles of 95°C for 10 sec, and 60°C for 45 sec. The KAPA SYBR FAST quantification product is representative of the template DNA (NGS library), whereas the quantification products generated with wild-type enzymes are not.
High consistency and product quality with a proven track record
Use of single DNA standard for all library types
Pre-diluted standards with very high lot-to-lot consistency
View data showing high consistency in quantification across library types and consistent amplification efficiency by KAPA Library Quantification Kits.
Figure 4. High consistency across different library types and across hundreds of production lots. Nine human WGS libraries (41% GC) and two microbial WGS libraries (Rhodococcus sp., 70% GC and Staphylococcus sp., 35% GC) were quantified with distinct lots (Lots 1 and 2), and distinct sets of reagents from the same lot (Set 1 and 2) of KAPA Library Quantification Kit for the Illumina platform.
Figure 5. High consistency across different library types and across hundreds of production lots. Amplification efficiencies for 56 consecutive lots of KAPA DNA Standards released since September 2009.
Figure 7. Accurate and sensitive quantification enables a uniform distribution of reads in muliplexed sequencing pools. Twenty-four indexed libraries, prepared from high-quality human genomic DNA (darker bars) or FFPE DNA (lighter bars), were quantified by qPCR using the KAPA Library Quantification Kit for Illumina platforms and combined to create three sequencing pools of equimolar concentration. Each pool was sequenced in a different lane of the same flow cell on a HiSeq 2500 instrument. (Figure 7) The twenty-four individual libraries represented a ~44-fold range of pre-pooling concentrations (5.2 nM to 229.8 nM), whereas the sequencing read distribution (Figure 8) only varied between 9.6% and 13.9%. The coefficient of variation (CV) for pre-pooling library concentration was 94.9%, 49.4%, and 127.9% for the libraries pooled for sequencing in lanes 1, 2, and 3, respectively. The KAPA Library Quantification Kit enabled accurate equimolar pooling, reducing the CV to 2.5%, 4.8%, and 11.2% after normalization.
Figure 8. Accurate and sensitive quantification enables a uniform distribution of reads in muliplexed sequencing pools. Twenty-four indexed libraries, prepared from high-quality human genomic DNA (darker bars) or FFPE DNA (lighter bars), were quantified by qPCR using the KAPA Library Quantification Kit for Illumina platforms and combined to create three sequencing pools of equimolar concentration. Each pool was sequenced in a different lane of the same flow cell on a HiSeq 2500 instrument. (Figure 7) The twenty-four individual libraries represented a ~44-fold range of pre-pooling concentrations (5.2 nM to 229.8 nM), whereas the sequencing read distribution (Figure 8) only varied between 9.6% and 13.9%. The coefficient of variation (CV) for pre-pooling library concentration was 94.9%, 49.4%, and 127.9% for the libraries pooled for sequencing in lanes 1, 2, and 3, respectively. The KAPA Library Quantification Kit enabled accurate equimolar pooling, reducing the CV to 2.5%, 4.8%, and 11.2% after normalization.
Complete kits include KAPA SYBR FAST qPCR Master Mix (2X), Primer Premix (10X) and a set of 6 DNA Standards. Reagents for qPCR (KAPA SYBR FAST qPCR Master Mix and Primer Premix) and DNA Standards (1–6) are also sold separately. Where noted, Master Mixes contain instrument-specific reference dyes, while the Universal kit includes ROX High and ROX Low (both 50X) separately.
