KAPA Library Preparation Kits

The KAPA Library Preparation Kit provides all of the enzymes and reaction buffers required for constructing Illumina libraries from fragmented dsDNA. Reaction buffers are supplied in convenient, concentrated “master mix” formats comprising all of the required reaction components except oligonucleotide adaptors or PCR primers. Similarly, a single enzyme mixture is provided for each step of the library construction process, reducing the number of pipetting steps. KAPA Library Preparation Kits contain the following modules:

1. End repair

The End Repair module produces blunt-ended, 5’-phosphorylated DNA fragments.

• End Repair Enzyme Mix containing T4 DNA Polymerase and T4 Polynucleotide Kinase (50 μL or 250 μL)
• 10X End Repair Buffer with dNTPs (100 μL or 500 μL)

2. A-Tailing

The A-Tailing module adds dAMP to the 3’-ends of the dsDNA fragments.

• A-Tailing Enzyme: Klenow Fragment 3’- 5’ exo- (30 μL or 150 μL)
• 10X A-Tailing Buffer (50 μL or 250 μL)

3. Adaptor ligation

The Ligation module ligates dsDNA adaptors with 3’-dTMP overhangs to library fragments.

• DNA Ligase (50 μL or 250 μL)
• 5X Ligation Buffer (100 μL or 500 μL)

4. Library amplification

High fidelity PCR is used to selectively enrich library fragments carrying appropriate adaptor sequences and to amplify the amount of DNA prior to sequencing. During PCR enrichment of libraries, it is critical that amplification bias is kept to a minimum to ensure uniform sequence coverage. This enables efficient sequencing where the highest overall coverage can be achieved from the least amount of total sequence.

KAPA HiFi DNA Polymerase is designed for low bias, high fidelity PCR representing the method of choice for the amplification of next-generation sequencing libraries. The intrinsic high processivity of the enzyme results in significant improvements in yield, sensitivity, speed, target length and the ability to amplify difficult templates. These enhancements result in lower amplification bias which provides more uniform sequence coverage. KAPA Library Preparation Kits are supplied with either a standard PCR library amplification module or a real-time PCR amplification module. Both modules are supplied as a 2X master mix and contain the novel KAPA HiFi DNA Polymerase, engineered for high fidelity and processivity and capable of balanced amplification of complex library DNA. KAPA Real-Time PCR Library Amplification Kit also includes SYBR® Green I dye and four fluorescent standards to allow for real-time monitoring of library amplification.

End-Point PCR module: 2X KAPA HiFi HotStart ReadyMix (250 μL or 1250 μL)

OR

Real-time PCR module: 2X KAPA HiFi HotStart Real-Time PCR Master Mix (250 μL or 1250 μL). 4 x Fluorescent Standards (1500 μL each)

The KAPA Real-Time PCR Library Amplification module is designed to address bias resulting from the DNA polymerase and over-amplification. Real-time monitoring of library amplification provides additional information required to optimize the number of amplification cycles and minimize over-amplification. The benefits of performing high fidelity, real-time PCR for next-generation sequencing library amplification include:

• Real-time monitoring of amplification allows precise control over the optimal number of PCR cycles.
• Real-time amplification workflows are amenable to automation.
• Real-time amplification plots provide quality metrics for individual enriched libraries, eliminating expensive and time- consuming post-enrichment gel electrophoresis and identifying inconsistencies in library preparation.
• Seamless integration with KAPA Library Quantification Kits.

KAPA Library Preparation Kits with Real-Time Library Amplification module contain KAPA HiFi HotStart Real-Time PCR Master Mix (2X), a ready-to-use cocktail containing all components for PCR, except primers and template. The 2X Master Mix contains KAPA HiFi HotStart DNA Polymerase in a proprietary reaction buffer, dNTPs, MgCl2 (2.5 mM at 1X), SYBR® Green I dye and stabilizers. Four fluorescent standards are supplied, and are used to define a window for optimal amplification.

Product Applications

This kits are primarily intended for the construction of the following types of Illumina libraries, but may be used for other applications requiring efficient end-repair, A-tailing, ligation, and/or library amplification steps:

• Genomic DNA libraries.
• Paired-end DNA libraries.
• Paired-end multiplexed (indexed/barcoded) DNA libraries.

Ultra pure, high quality library construction reagents result in higher yield of adaptor-ligated library molecules

Sheared genomic DNA from three organisms (S. aureus, E. coli, or M.tuberculosis) was prepared in bulk by nebulization and 1 μg of identical starting material was used for each library. Libraries were constructed using the KAPA Library Preparation Kit and recommended protocol (green; 9 libraries), or using the Illumina TruSeq™ DNA Sample Prep Kit and Low-Throughput Protocol (orange; 15 libraries). Libraries were quantified by qPCR before size selection using the KAPA Library Quantification Kit according to recommended protocol. The estimated percentage of starting material that was converted to useful, adaptor-ligated (PCR-amplifiable) library molecules is provided. The KAPA Library Preparation Kit and protocol produced ~25-fold more adaptor-ligated library fragments from the same amount of starting material than did the standard Illumina Truseq™ DNA Sample Prep Kit.

An engineered, high fidelity DNA polymerase reduces amplification bias and improves sequencing coverage

Indexed libraries were constructed and amplified from 100 ng human genomic DNA using either the KAPA Library Preparation Kit (right) or the standard library preparation reagents and protocol in use at The Broad Institute (left). Individual libraries were quantified using qPCR, pooled prior to denaturation, and sequenced (pair-ended, 2 x 25 bp) on an Illumina HiSeq 2000. Libraries prepared using the KAPA Library Preparation Kit resulted in more uniform coverage distribution across the range of GC-content. Data courtesy of The Broad Institute.