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产品说明书: KAPA Hyper Prep C (96rxn)
Published: Nov 1, 2019 DOI: 10.21769/BioProtoc.1010523 Views: 2205
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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-tailin1.1Assemble each end repair and A-tailing reaction in a tube or well of a PCR plate as follows:
*The buffer and enzyme mix should preferably be pre-mixed and added in a single pipetting step. Premixes are stable for ≤ 24 hrs at room temperature, for ≤ 3 days at 2 °C to 8 °C, and for ≤ 4 weeks at -15 °C to -25 °C.1.2Vortex gently and spin down briefly. Return the plate/tube(s) to ice. Proceed immediately to the next step.1.3Incubate in a thermocycler programmed as outlined below:
*A heated lid is required for this incubation. If possible, set the temperature of the lid at 85 °C, instead of the usual ~105 °C.
**If proceeding to the adapter ligation reaction setup without any delay, the reaction may be cooled to 20 °C instead of 4 °C.
1.4Proceed immediately to Adapter Ligation (step 2). - Adapter Ligation2.1Dilute adapter stocks to the appropriate concentration, as outlined in Table 2 (p. 5).2.2In the same plate/tube(s) in which end repair and A-tailing was performed, assemble each adapter ligation reaction as follows:
*The water, buffer and ligase enzyme should preferably be premixed and added in a single pipetting step. Premixes are stable for ≤ 24 h at room temperature, for ≤ 3 days at 4 °C, and for ≤ 4 weeks at -20 °C.2.3Mix thoroughly and centrifuge briefly.2.4Incubate at 20 °C for 15 min.
Note: to achieve higher conversion rates and library yields, particularly for low-input samples, consider increasing the ligation time – to a maximum of 4 h at 20 °C, or overnight at 2 °C to 8 °C. Please note that longer ligation times may lead to increased levels of adapter-dimer. Adapter concentrations may have to be optimized if ligation times are extended significantly.2.5Proceed immediately to the next step. - Post-ligation Cleanup3.1In the same plate/tube(s), perform a 0.8X bead- based cleanup by combining the following:
3.2Mix thoroughly by vortexing and/or pipetting up and down multiple times.3.3Incubate the plate/tube(s) at room temperature for 5-15 min to bind DNA to the beads.3.4Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.3.5Carefully remove and discard the supernatant.3.6Keeping the plate/tube(s) on the magnet, add 200 µL of 80% ethanol.3.7Incubate the plate/tube(s) on the magnet at room temperature for ≥ 30 sec.3.8Carefully remove and discard the ethanol.3.9Keeping the plate/tube(s) on the magnet, add 200 µL of 80% ethanol.3.10Incubate the plate/tube(s) on the magnet at room temperature for ≥ 30 sec.3.11Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.3.12Dry the beads at room temperature for 3-5 min, or until all of the ethanol has evaporated.
Caution: over-drying the beads may result in reduced yield.3.13Remove the plate/tube(s) from the magnet.3.14Thoroughly resuspend the beads:
•in 25 µL of elution buffer (10 mM Tris-HCl, pH 8.0-8.5) to proceed with Library Amplification (step 4), or
•in 55 µL of elution buffer (10 mM Tris-HCl, pH 8.0-8.5) to proceed with double-sided size selection (Appendix 1).3.15Incubate the plate/tube(s) at room temperature for 2 min to elute DNA off the beads.3.16Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.3.17Transfer the clear supernatant to a new plate/tube(s):
•to proceed with Library Amplification (step 4), transfer 20 µL of supernatant, or
•to proceed with double-sided size selection (Appendix 1), transfer 50 µL of supernatant. - 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.
4.1Assemble each library amplification reaction as follows:
*Or another, suitable 10X library amplification primer mix. The recommended final concentration of each primer in the library amplification reaction is 0.5-4 µM. Also refer to Important Parameters: Library Amplification (p. 6).4.2Mix thoroughly and centrifuge briefly.4.3Amplify using the following cycling protocol:
*Optimization of the annealing temperature may be required for non- standard (i.e., other than Illumina TruSeq) adapter/primer combinations.4.4Proceed directly to Post-amplification Cleanup (step 5). - Post-amplification Cleanup5.1In the library amplification plate/tube(s) perform a 1X bead-based cleanup by combining the following:
5.2Mix thoroughly by vortexing and/or pipetting up and down multiple times.5.3Incubate the plate/tube(s) at room temperature for 5-15 min to bind DNA to the beads.5.4Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.5.5Carefully remove and discard the supernatant.5.6Keeping the plate/tube(s) on the magnet, add 200 µL of 80% ethanol.5.7Incubate the plate/tube(s) on the magnet at room temperature for ≥ 30 sec.5.8Carefully remove and discard the ethanol.5.9Keeping the plate/tube(s) on the magnet, add 200 µL of 80% ethanol.5.10Incubate the plate/tube(s) on the magnet at room temperature for ≥ 30 sec.5.11Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.5.12Dry the beads at room temperature for 3-5 min, or until all of the ethanol has evaporated.
Caution: over-drying the beads may result in reduced yield.5.13Remove the plate/tube(s) from the magnet.5.14Thoroughly resuspend the beads in an appropriate volume of elution buffer (10 mM Tris-HCl, pH 8.0-8.5) or PCR-grade water. Always use PCR-grade water if proceeding to target capture. Note: If proceeding with a second post-amplification cleanup, or double-sided size selection (Appendix 1), resuspend the beads in 55 µL of elution buffer.5.15Incubate the plate/tube(s) at room temperature for 2 min to elute DNA off the beads.5.16Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.5.17Transfer the clear supernatant to a new plate/tube(s) and proceed with size selection (refer to Appendix 1), library QC, target capture or sequencing, as appropriate. Store purified, amplified libraries at 2 °C to 8 °C for 1-2 weeks, or at -15 °C to -25 °C.
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Product documentation > Product manual > KAPA NGS
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