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KAPA Library Quantification

Next generation DNA sequencing meets next generation qPCR.

	qPCR specifically quantifies only PCR-competent DNA molecules,
	is highly sensitive allowing accurate quantification of low concentration libraries,
	is amenable to automated liquid handling.

KAPA Library Quantification Kits are optimized for the Illumina Genome Analyzer, Roche 454 Titanium series, and Roche 454 FLX series platforms and include defined, reliable DNA concentration standards and state-of-the-art qPCR reagents, containing a DNA polymerase engineered for SYBR® Green-based qPCR through a process of molecular evolution.

Library quantification

qPCR library quantification results in streamlined workflows

KAPA Library Quantification Kits eliminate the need for time-consuming and expensive titrations and provide a conducive format for streamlining high-throughput workflows.

Reliable quantification results in consistent cluster density

"Before qPCR was adopted for library quantification, cluster density was extremely variable. Implementation of the KAPA Library Quantification Kit in our sequencing workflows resulted in a significant reduction in variability across multiple libraries, negating the need for cluster amplification titration runs."

- Broad Institute, Cambridge, MA U.S.A.

Fig.1 Cluster density before and after implementation of the KAPA Library Quantification Kit (right). The implementation of KAPA Library Quantification Kits into the Illumina GA sequencing workflow at the Broad Institute significantly reduced cluster density variability and eliminated the need for titrations. Average number of clusters per tile are shown for consecutive libraries.

Efficient amplification of a wide range of templates during qPCR

Traditional qPCR reagents are optimized for short amplification targets; longer targets, unbalanced GC-content, and prob- lematic secondary structures may result in low amplification efficiency and unreliable quantification of some library molecules. To address the demands of quantifying complex DNA libraries, Kapa Biosystems has engineered a DNA polymerase specifi- cally for SYBR® Green-based qPCR, enabling efficient amplification of targets that present a challenge to wild-type enzymes. KAPA Library Quantification Kits contain this engineered polymerase to ensure robust amplification of longer fragments, across a broad range of GC-content, required for accurate library quantification.

Fig.2 Fragment size distributions before and after qPCR. Fragment size distributions before (grey fill) and after qPCR amplification using three commercial qPCR master mixes (KAPA SYBR® FAST (blue), Competitor S (red), and Competitor F (orange)). Competitor kits contained wild-type Taq polymerase. Reactions were performed with the following cycling protocol: 95 ºC for 10 min followed by 40 cycles of 95 ºC for 10 sec and 60 ºC for 45 sec.

Fig.3 Robust amplification translates into accurate qPCR quantification of diverse libraries. The KAPA Libary Quantificaiton Kit was used to determine the concentration of two Illumina GA libraries with unusual GC content (Rhodococcus sp.; ~70% GC - shown above, Staphylococcus sp.; ~35% GC - not shown). Both libraries amplified with efficiency >95%. Two-fold dilution series (1:1000 through 1:16000) were prepared in triplicate, and qPCR performed according to the recommendations in the product technical data sheet.

Reliable DNA quantification standards with minimal variability from lot-to-lot

Fig. 4 Lot-to-lot variability of the KAPA Library Quantification Kit for the Roche Titanium series platform. Three distinct lots (red, pink, blue) were compared by analyzing amplification plots of each set of quantification standards. Triplicates of each data point were averaged.

Fig. 5 Minimal lot-to-lot and kit-to-kit variability. 9 human DNA libraries and two microbial DNA libraries were used to compare quantification results obtained with distinct lots ("Lot 1" and "Lot 2"), and distinct sets of reagents from the same lot ("set 1" and "set 2") of KAPA Library Quantification Kits for the Illumina GA platform.
 

*All kits contain 5 mL KAPA SYBR® FAST qPCR Master Mix (2X), 1 mL Primer Premix, and 6 x 80 uL DNA Quantification Standards. Kits contain primers, DNA standards, and qPCR reagents specific for both DNA sequencing platform and qPCR instrument. Primer Premix and DNA Quantification Standards are also sold separately.

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