KAPA Hyper Prep Kits

Shift your workflow into hyperdrive.

The KAPA Hyper Prep Kit is a versatile, streamlined solution for DNA library preparation for Illumina® sequencing.

The novel one-tube chemistry, contain optimally formulated and evolved enzymes that enable higher yields of adapter-ligated library and lower amplification bias. This translates to higher library diversity, lower duplication rates and more uniform coverage, particularly for FFPE and low-input samples.

NEW! KAPA Single-Indexed Adapter Kits are now available. For more information on the appropriate Adapter Kit for your library construction workflow and input, scroll down to the Ordering section, or download the KAPA Single-Indexed Adapter Calculator.

Download our KAPA Single-Indexed Adapter Kit Calculator

 

For Research Use Only. Not for use in diagnostic procedures.

Product Highlights

Improve library yields and sequence quality

  • Higher library yields translate to greater molecular complexity
  • Fewer cycles of amplification with KAPA HiFi DNA Polymerase results in lower duplication rates and improved coverage

Create high-quality libraries from FFPE samples

  • Generate high-quality libraries from 250 ng FFPE DNA or less*
  • Significantly lower duplication rates and higher coverage*
The streamlined KAPA Hyper Prep workflow. End repair, A-tailing and ligation is performed in a single tube, without intervening cleanup steps. The workflow is ideally suited for robust, high-throughput sample preparation for Illumina sequencing. Data on file.

Complete library construction in less than 3 hours

  • One-tube workflow with minimal cleanup steps reduces overall time, and minimizes hands-on time
  • Sample-to-library in <2 hours for PCR-free workflows, or <3 hours with amplification*
  • Fewer handling steps lead to improved consistency and reproducibility

Improve performance with low-input samples

  • Generate more sequence-quality library molecules without increasing adapter-dimers
  • Increase yields without inducing size bias

 

*Data on file.

Related Products

Are you sequencing low-input, FFPE or high quality DNA?  RNA?  Check out these Kapa NGS products to improve your workflow and results:

Sample QC

KAPA hgDNA Quantification and QC Kit

Library Amplification

KAPA Library Amplification Kits

Library Quantification

KAPA Library Quantification Kits

KAPA Stranded mRNA-Seq Kits

KAPA Stranded mRNA-Seq Kits

Applications
  • Whole Genome Sequencing
  • Whole Exome Sequencing
  • Targeted Sequencing (custom panels)
  • ChIP-seq
  • Amplicon Sequencing
  • Methyl-seq
Kit Specifications and Contents / Storage

Kits can be stored for up to 12 months at -20˚C.

Kits contain KAPA End Repair & A-Tailing Buffer, KAPA End Repair & A-Tailing Enzyme, KAPA Ligation Buffer, KAPA DNA Ligase, KAPA HiFi HotStart ReadyMix and KAPA Library Amplification Primer Mix

Components

Hyper_Component Chart

Specifications

Spec
Description
Compatible Platform
llumina HiSeq, MiSeq, NextSeq and GAIIx
Library Type
DNA
Starting Material
Fragmented DNA, cDNA, or PCR amplicons
Input Amount
1 ng–1 µg
FAQs
What are the input requirements?

This protocol has been validated for library construction from 1 ng–1 μg of appropriately fragmented dsDNA. However, libraries can be prepared from lower input amounts if the sample represents sufficient copies to ensure the requisite coverage and complexity in the final library.

What are the major steps in library construction?

  • 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 library fragments
  • Library amplification (optional), which employs high-fidelity, low-bias PCR to amplify library fragments carrying appropriate adapter sequences on both ends

Are there safe stopping points in the library construction process?

The library construction process, from end repair and A-tailing to final, amplified library, can be performed in less than 3 hours, depending on experience and the number of samples being processed. If necessary, the protocol may be safely paused after completion of the post-ligation cleanup, or prior to post-amplification cleanup.

Adapter-ligated library may be stored at 4°C for one week, or at -20°C at least one month before amplification, target enrichment and/or sequencing. To avoid degradation, always store DNA in a buffered solution (10 mM Tris-HCl, pH 8.0), and minimize the number of freeze-thaw cycles.

What are your Covaris® shearing recommendations?

  • If you are performing a cleanup between shearing and end repair, shear in 10 mM Tris-HCl (pH 8 or 8.5) + 1 mMEDTA.
  • If you are not performing a cleanup between shearing and end repair, shear in 10 mM Tris-HCl (pH 8 or 8.5) + 0.1 mMEDTA.
  • Never shear DNA in water.

What adapters should I use with this kit and at what concentration?

KAPA Adapters are recommended for use with KAPA Hyper Prep Kits, except for methyl-seq applications. KAPA Hyper Prep Kits are also compatible with non-indexed, single-indexed, and dual-indexed adapters that are routinely used in Illumina TruSeq, Roche NimbleGen SeqCap EZ, Agilent SureSelect, and other similar library construction and target capture workflows. Custom adapters that are of similar design and are compatible with “TA-ligation” of dsDNA may also be used, remembering that custom adapter designs may impact library construction efficiency.

Ligation efficiency is robust for adapter-insert molar ratios from 10:1 to >200:1. The recommended adapter concentrations for different inputs are given in the table below. Note that high adapter-insert molar ratios are beneficial for low-input and challenging samples. When optimizing worfklows for DNA inputs ≤25 ng, it is recommended that two or three adapter concentrations be evaluated. Try the recommended concentration in the table, as well as one or two additional concentrations in the range that is 2 – 10 times higher.

Input DNA Adapter stock concentration Adapter:insert
molar ratio
1 μg 15 μM 10:1
500 ng 15 μM 20:1
250 ng 15 μM 40:1
100 ng 15 μM 100:1
50 ng 15 μM 200:1
25 ng 7.5 μM 200:1
10 ng 3 μM 200:1
5 ng 1.5 μM 200:1
2.5 ng 750 nM 200:1
1 ng 300 nM 200:1

High Concentration (30 µM) KAPA Adapter Kits are recommended for library construction from 5 ng – 1 µg inputs, whereas Low Concentration (1.5 µM) KAPA Adapter Kits are recommended for inputs <5 ng. For assistance with adapter compatibility and ordering, please visit kapabiosystems.com/support.

Where do I find more information about KAPA Adapters?

Please refer to the KAPA Single-Indexed Adapter Technical Data Sheet for information about barcode sequences, pooling, kit configurations, formulation, and dilution of KAPA Single-Indexed Adapters.

What QC testing is performed on KAPA Adapters?

KAPA Adapters undergo extensive qPCR- and sequencing-based functional and QC testing to confirm:

  • optimal library construction efficiency
  •  minimal levels of adapter-dimer formation
  • nominal levels of barcode cross-contamination

Library construction efficiency and adapter-dimer formatin are assessed in a low-input library construction workflow. The conversion rate achieved in the assay indicates library construction efficiency. This is calculated by measuring the yield of adapter-ligated library (before any amplification) by qPCR (using the KAPA Library Quantification Kit), and expressing this as a % of input DNA. To assess adapter-dimer formation, a modified library construction protocol— designed to measure adapter dimer with high sensitivity—is used. Pass criteria for this assay translate to adapter-dimer carry-over in a standard workflow in the range of 0 – 2 %.

Barcode cross-contamination is assessed by sequencing. Each adapter is ligated to a unique, synthetic insert of known sequence, using a standard library construction protocol. These constructs pooled and sequenced on a MiSeq. For every barcode, the number of reads (in the range of 115,000 – 500,000) associated with each insert is counted, and the total % correct inserts calculated. Contamination of any barcode with any other single barcode is guaranteed to be <0.25%. The total level of contamination for any barcode is typically in the range of 0.1 – 0.5%. This assay is unable to distinguish between chemical cross-contamination and adapter “cross-talk”, and measures the total number of incorrect inserts resulting from both phenomena.

Are there specific workflow recommendations for FFPE samples?

No, follow the standard protocol to construct FFPE libraries for target capture. For WGS workflows, a size selection step will most likely be required.

What is the proportion of fragmented DNA that is converted to adapter-ligated molecules?

The proportion of fragmented DNA that is successfully converted to adapter-ligated molecules decreases as input is reduced. When starting library construction (end repair and A-tailing) with >10 ng fragmented genomic DNA, 5%–35% of input DNA is typically recovered as adapter-ligated molecules. Workflows that contain additional bead-based cleanups or size selection prior to library amplification are likely to result in a lower yield of adapter-ligated molecules.

The novel, one-tube KAPA Hyper Prep chemistry leads to less adapter-dimer formation and carry-over. A single bead-based cleanup after adapter ligation is sufficient to remove unused adapter and adapter dimer, even at the high adapter:insert molar ratios recommended for low-input applications.

Does the protocol support size selection?

If required, any commonly used size selection technique (e.g., the double-sided or an electrophoresis-based method) may be integrated in this protocol.

Size selection may be carried out at alternative points in the overall workflow, for example:

  • prior to end repair of fragmented DNA,
  • after the post-ligation cleanup, or
  • after library amplification.

The standard KAPA Hyper Prep protocol does not include size selection. Detailed protocols for double-sided size selection prior to library construction, after ligation or after library amplification may be found in Appendix 1 of the KAPA Hyper Prep Technical Data Sheet.

Which enzyme is used for amplification?

KAPA Hyper Prep Kits contain KAPA HiFi HotStart ReadyMix for library amplification. This contains the novel, B-family HiFi HotStart DNA Polymerase, which was evolved for low-bias, high-efficiency, high-fidelity PCR. KAPA HiFi has become the gold standard for NGS library amplification (1, 2, 3, 4).

  1. Oyola, S.O. et al. BMC Genomics 13, 1 (2012)
  2. Quail M.A. et al. Nature Methods 9, 10-11 (2012)
  3. Quail M.A. et al. BMC Genomics 13, 341 (2012)
  4. Ross, M.G. et al. Genome Biology 14: R51 (2013)

How many cycles of library amplification should be used?

If cycled to completion (not recommended) a single 50 µL KAPA HiFi library amplification reaction can produce 8–10 µg of amplified library. To minimize over-amplification and associated undesired artefacts, the number of amplification cycles should be tailored to produce the optimal amount of amplified library required for downstream processes. This is typically in the range of 250 ng–1.5 µg of final, amplified library.

Quantification of adapter-ligated libraries prior to library amplification can greatly facilitate the optimization of library amplification parameters, particularly when a library construction workflow is first established or optimized. The amount of template DNA (adapter-ligated molecules) available for library amplification may be determined using the KAPA Library Quantification Kit. A simple algorithm can subsequently be used to theoretically predict the number of amplification cycles needed to achieve a specific yield of amplified library. A calculator that can be used for purpose is available on request from kapabiosystems.com/support.

The estimated number of amplification cycles for libraries prepared from different amounts of input DNA is given in the table below. The actual optimal number of amplification cycles may be 1–3 cycles higher or lower, depending on the sample type and size distribution of the input DNA.

Input DNA1 Number of cycles required to generate:
100 ng 1 μg
1 μg 0–1 2–3
500 ng 0–1 3–4
250 ng 1–3 4–6
100 ng 3–4 6–7
50 ng 4–5 7–8
25 ng 5–7 8–10
10 ng 8–11 11–14
5 ng 10–13 13–16
2.5 ng 12–14 15–17
1 ng 14–16 17–19

1Input into end repair and A-tailing reaction

Is library amplification mandatory?

If the amount of adapter-ligated library retrieved after the post-ligation cleanup is sufficient for the next process (e.g. direct sequencing), and the library molecules contain all the adapter motifs needed for that process, library amplification may be omitted. This further streamlines the workflow and reduces the overall library preparation time to <2 hours. The high conversion efficiency achievable with the KAPA Hyper Prep Kit enables PCR-free workflows from as little as 100 ng input DNA. KAPA Hyper Prep Kits without amplification reagents (KK8501, KK8503 and KK8505) are available for PCR-free workflows.

Is the KAPA Library Amplification Primer Mix compatible with all Illumina libraries?

KAPA Hyper Prep Kits contain a new, highly optimized Library Amplification Primer Mix, designed to eliminate or delay primer depletion during library amplification reactions performed with KAPA HiFi DNA polymerase. The primers in the mix are based on the P5 and P7 Illumina flow cell sequences, and are suitable for the amplification of libraries prepared with full-length adapters. User-supplied primer mixes may be used in combination with incomplete or custom adapters. Please contact support@kapabiosystems.com for guidelines on the formulation of user-supplied library amplification primers.

How do I know if I over-amplified my library?

In library amplification reactions, primers are typically depleted before dNTPs.  When DNA synthesis can no longer take place due to substrate depletion, subsequent rounds of DNA denaturation and annealing result in the separation of complementary DNA strands, followed by imperfect annealing to non-complementary partners.  This presumably results in the formation of so-called “daisy chains” or “tangled knots”, comprising large assemblies of improperly annealed, partially double-stranded, heteroduplex DNA.  These species migrate slower and are observed as secondary, higher molecular weight peaks during the electrophoretic analysis of amplified libraries.

What are the consequences of over-amplification?

Excessive library amplification can result in unwanted artifacts such as PCR duplicates, chimeric library inserts, amplification bias and heteroduplex formation. It is generally best to limit the extent of library amplification as much as possible, while ensuring that sufficient material is generated for QC and sequencing.

How do I assess the quality of my library after preparation?

Library size distribution, and the absence of primer dimers and/or over-amplification products, should be confirmed by means of an electrophoretic method. KAPA Library Quantification Kits are recommended for qPCR-based quantification of libraries prior to pooling for target capture or sequencing. qPCR-based quantification of adapter-ligated libraries (prior to library amplification) is not routinely done, but can provide useful data for protocol optimization and troubleshooting.

Ordering

Kits contain KAPA End Repair and A-tailing Enzyme Mix, End Repair and A-tailing Buffer, DNA Ligase, Ligation Buffer, KAPA HiFi HotStart ReadyMix (2X), and KAPA Library Amplification Primer Mix (10X).

Kit Code
Roche Cat. No
Description
Kit Size
How to buy
KK8501
07962339001
Without amplification module
8 reactions
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KK8503
07962355001
Without amplification module
24 reactions
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KK8505
07962371001
Without amplification module
96 reactions
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KK8500
07962312001
With KAPA Library Amplification Primer Mix (10X)
8 reactions
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KK8502
07962347001
With KAPA Library Amplification Primer Mix (10X)
24 reactions
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KK8504
07962363001
With KAPA Library Amplification Primer Mix (10X)
96 reactions
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Accessories

KK8700, KK8701 and KK8702 are recommended for 5 ng – 1µg inputs, whereas KK8710, KK8711 and KK8712 are recommended for
inputs <10 ng.  Adapter Set A contains indices 2, 4, 5, 6, 7, 12, 13, 14, 15, 16, 18 and 19. Adapter Set B contains indices 1, 3, 8, 9, 10, 11, 20, 21, 22, 23, 25, 27. All kits contain KAPA Adapter Dilution Buffer.

Kit Code
Roche Cat. No
Description
Kit Size
How to buy
KK8000
07983271001
KAPA Pure Beads (5 mL)
5 mL
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KK8001
07983280001
KAPA Pure Beads (30 mL)
30 mL
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KK8002
07983298001
KAPA Pure Beads (60 mL)
60 mL
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KK8700
08005699001
KAPA Single-Indexed Adapter Kit, Set A + B (30 µM)
24 adapters x 40 µl each
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KK8701
08005702001
KAPA Single-Indexed Adapter Kit, Set A (30 µM)
12 adapters x 40 µl each
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KK8702
08005729001
KAPA Single-Indexed Adapter Kit, Set B (30 µM)
12 adapters x 40 µl each
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KK8710
08005770001
KAPA Single-Indexed Adapter Kit, Set A + B (1.5 µM)
24 adapters x 40 µl each
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KK8711
08005788001
KAPA Single-Indexed Adapter Kit, Set A (1.5 µM)
12 adapters x 40 µl each
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KK8712
08005796001
KAPA Single-Indexed Adapter Kit, Set B (1.5 µM)
12 adapters x 40 µl each
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