KAPA Library Quantification Kits

Ensuring accurate Next-Generation Sequencing (NGS) results requires precise library quantification. The gold standard for this is qPCR, which measures only sequencing-competent fragments, providing all reagents, standard curves for various library types, and essential data analysis support for successful sequencing runs. Our complete library solution offers:

• 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
• Data analysis templates and assistance available from sequencing support 

Figure 1: 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 minutes, followed by 40 cycles of 95°C for 10 seconds, and 60°C for 45 seconds. 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.

KAPA Library Quantification Kits contain KAPA SYBR FAST DNA Polymerase, engineered through our directed evolution technology to amplify diverse DNA fragments with similar efficiency. This is important for the amplification of the heterogeneous populations that make up NGS libraries. Library quantification kits containing wild-type DNA polymerases only count “easy” library molecules.¹

The use of a single, highly consistent set of DNA standards simplifies the sequencing workflow, eliminating the need for separate standards for different library types. Together with pre-diluted standards with very high lot-to-lot consistency, this ensures accurate and reproducible qPCR-based quantification across all library construction methods.

Figure 2: 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.

Figure 3: 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 the KAPA Library Quantification Kit.

Figure 4: 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.

A key factor in high-quality library preparation is achieving optimal cluster densities to maximize sequencing output. This is especially challenging when working with low-quality samples, like DNA from FFPE tissues, which are often degraded. Specialized protocols and kits are essential to overcome these challenges and ensure accurate, high-quality results. We provide:

• Optimal and predictable cluster densities to maximize sequencing capacity and throughput
• Reliable results for all library types, including from low-quality FFPE DNA

Figure 5: Accurate and reproducible library quantification enables improved and consistent cluster densities.

The implementation of KAPA Library Quantification Kit into the Illumina® GA sequencing workflow at The Broad Institute (Cambridge, MA, USA) significantly reduced cluster density variability and eliminated the need for titrations. Average number of clusters per tile are shown for consecutive libraries.

Equimolar pooling ensures that each indexed library contributes equally to the final pool, regardless of its initial concentration. This strategy maximizes data quality and consistency for every sample. Our kits ensure:

• Equimolar pooling of indexed libraries, irrespective of pre-pooling library concentrations
• Uniform distribution of reads across all the libraries in a pool

Figure 6: Accurate and sensitive quantification enables a uniform distribution of reads in multiplexed 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. The twenty-four individual libraries represented a ~44-fold range of pre-pooling concentrations (5.2 nM to 229.8 nM) (Figure 6A), whereas the sequencing read distribution only varied between 9.6% and 13.9% (Figure 6B). 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.