Product Description
The KAPA Hyper Prep Kit provides a versatile, streamlined protocol for the rapid construction of libraries for Illumina sequencing from fragmented, double-stranded DNA (dsDNA). The novel chemistry and streamlined, one-tube protocol improves the efficiency and consistency of library construction across a wide range of sample types and inputs.
The workflow combines enzymatic steps and employs minimal bead-based cleanups, thereby reducing sample handling and overall library preparation time to 2-3 h. The kit contains all of the enzymes and reaction buffers required for:
- end repair and A-tailing, which produces end-repaired, 5'-phosphorylated, 3'-dA-tailed dsDNA fragments;
- adapter ligation, during which dsDNA adapters with 3'-dTMP overhangs are ligated to 3'-dA-tailed molecules;
- library amplification (optional), which employs high- fidelity, low-bias PCR to amplify library fragments carrying appropriate adapter sequences on both ends.
The kit provides a single, concentrated buffer and a single enzyme mixture for each of the two library construction steps. This offers the best combination of product stability, convenience and efficiency. Adapters and beads required for cleanups after adapter ligation and library amplification are not included. KAPA Pure Beads and KAPA Adapters are sold separately.
In order to maximize sequence coverage uniformity, it is critical to minimize library amplification bias. KAPA HiFi DNA Polymerase is designed for low-bias, high- fidelity PCR, and is the reagent of choice for NGS library amplification1, 2, 3, 4.
KAPA Hyper Prep Kits include KAPA HiFi HotStart ReadyMix (2X), a ready-to-use PCR mix comprising all the components for library amplification – except primers and template. Kits also include Library Amplification Primer Mix (10X), designed for the high- efficiency amplification of Illumina libraries flanked by adapters containing the P5 and P7 flow cell sequences.
- Oyola, S.O., et al., BMC Genomics 13, 1 (2012).
- Quail, M.A., et al., Nature Methods 9, 10 (2012).
- Quail, M.A., et al., BMC Genomics 13, 341 (2012).
- Ross, M.G., et al., Genome Biology 14, R51 (2013).
Product Applications
KAPA Hyper Prep Kits are ideally suited for low- and high-throughput NGS library construction workflows that require end repair, A-tailing, adapter ligation and library amplification (optional). Kits are designed for library construction from a wide range of sample types and inputs (1 ng-1 µg), and are compatible with complex genomic DNA, cell-free/circulating tumor DNA and low- quality DNA such as FFPE samples. For small genomes, cell-free/circulating tumor DNA and lower complexity samples such as ChIP DNA, amplicons or cDNA (for RNA- seq), library construction from lower inputs (~100 pg or more) may be attempted.
The protocol is automation-friendly and may be incorporated into workflows for a wide range of NGS applications, including:
•whole-genome, shotgun sequencing
•whole exome or targeted sequencing, using Roche SeqCap EZ, Agilent SureSelect, Illumina TruSeq, or IDT xGen Lockdown Probes, or other hybridization capture systems
•ChIP-seq
•RNA-seq (starting with cDNA)
•methyl-seq (in combination with KAPA HiFi HotStart
•Uracil+ ReadyMix for library amplification).
Process Workflow
Library Construction Protocol
Note: This protocol does not include size selection. Please refer to Appendix 1 for detailed double-sided size selection protocols.
- End Repair and A-tailin
- Adapter Ligation
- Post-ligation Cleanup
- Library Amplification
Note: Please refer to Important Parameters: Library Amplification (pp. 6-7) and the KAPA NGS Library Preparation Technical Guide for more information on optimizing library amplification.
- Post-amplification Cleanup