KAPA HyperPrep Kits

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

Overview

KAPA HyperPrep Kits offer a streamlined library preparation protocol that combines several enzymatic steps and eliminates bead cleanups to reduce library preparation time and improve consistency. The single-tube chemistry offers improvements to library construction efficiency, particularly for challenging samples such as FFPE and cell-free DNA. KAPA HyperPrep Kits offer a complete library preparation solution with KAPA Adapters and KAPA HyperPure Beads.

For the ultimate NGS automation solution, the KAPA HyperPrep workflow is also available on our walk-away automated platform, the AVENIO Edge System.

Features and Benefits of KAPA HyperPrep Kits

Robust and reproducible library construction in 2-3 hours with high conversion rates and library complexity, particularly for FFPE
PCR-free workflows from lower sample inputs and improved sequence coverage for workflows with PCR
Compatibility with a wide range of sample types and inputs, and flexibility with respect to fragment size, adapter design and library amplification
Automation methods for various vendors available
Validated with the KAPA HyperCap and KAPA HyperPETE workflows

Figure 1: The KAPA HyperPrep Workflow

*KAPA Adapter and KAPA Cleanup Beads Kits sold separately.

Product highlights

Improved library yields and quality

Higher library yields translating to greater molecular complexity
Higher conversion of input DNA to adapter-ligated library
Fewer cycles of amplification with KAPA HiFi DNA Polymerase, resulting in lower duplication rates and improved coverage

View comparison data for higher conversion of input DNA to adapter-ligated library by KAPA HyperPrep Kit compared to Supplier I or Supplier N.

Figure 2. KAPA HyperPrep Kit enable higher library construction efficiency. Conversion rates for libraries prepared for target capture from different amounts of Covaris-sheared human genomic DNA, using the KAPA HyperPrep or Supplier library construction kits. Libraries were prepared according to manufacturer’s instructions. Input DNA was quantified by Qubit, whereas the qPCR-based KAPA Library Quantification Kit was used to determine KAPA HyperPrep and Suppliers' library yields after adapter ligation. Conversion rates for Supplier N libraries cannot be measured directly with the KAPA Library Quantification Kit and were derived from post-amplification yields.

Minimal amplification bias

Kits with or without an amplification module
Lower PCR bias resulting in more uniform coverage in PCR workflows

See performance data showing high coverage uniformity using KAPA HyperPrep Kits and minimal amplification bias with KAPA HiFi polymerase.

Figure 3. KAPA HiFi introduces minimal amplification bias and offers high coverage uniformity. GC bias plots for libraries prepared for whole-genome shotgun sequencing of bacteria with extreme genomic GC content (Clostridium, 29% GC and Bordetella, 68% GC). Libraries were prepared with the KAPA HyperPrep Kit from 100 ng or 1 ng DNA, Covaris-sheared to an average size of ~200 bp; and sequenced without amplification, or after amplification with KAPA HiFi. Minimal amplification bias was introduced by KAPA HiFi, despite a relatively high number of PCR cycles (12 cycles for Clostridium and 13 cycles for Bordetella). Paired-end sequencing (2 x 300 bp) was performed on an Illumina MiSeq, and data analyzed using Picard.

Figure 4. KAPA HiFi introduces minimal amplification bias and offers high coverage uniformity. GC bias plots for libraries prepared for whole-genome shotgun sequencing of bacteria with extreme genomic GC content (Clostridium, 29% GC and Bordetella, 68% GC). Libraries were prepared with the KAPA HyperPrep Kit from 100 ng or 1 ng DNA, Covaris-sheared to an average size of ~200 bp; and sequenced without amplification, or after amplification with KAPA HiFi. Minimal amplification bias was introduced by KAPA HiFi, despite a relatively high number of PCR cycles (12 cycles for Clostridium and 13 cycles for Bordetella). Paired-end sequencing (2 x 300 bp) was performed on an Illumina MiSeq, and data analyzed using Picard.

High coverage of FFPE samples

Higher library yield, higher library complexity and fewer PCR cycles
Lower duplication rates and higher coverage

View data showing higher library diversity, lower duplication rates and improved coverage and using KAPA HyperPrep Kit in comparison with Supplier I.

Figure 5. KAPA HyperPrep Kit enable improved coverage depth for FFPE samples. Key sequencing metrics for libraries prepared from 100 ng of FFPE DNA for target capture, using either the KAPA HyperPrep Kit or Supplier I. Captures were performed with the SeqCap EZ Comprehensive Cancer Design (4 Mb) according to the manufacturer’s instructions, with the exception that the number of pre-capture amplification cycles for each library type was optimized based on post-ligation yields (10 cycles for KAPA HyperPrep Kit vs. 14 cycles for Supplier I libraries). All libraries were amplified for 13 cycles after capture. More efficient library construction and less PCR bias with the KAPA HyperPrep Kit resulted in higher library complexity prior to capture, lower duplication rates, and improved coverage. Sequencing (2 x 75 bp) was performed on an Illumina HiSeq 2500. Sequencing reads were down-sampled to ~14 million per library prior to analysis with Picard.

Figure 6. KAPA HyperPrep Kit enable improved coverage depth for FFPE samples. Key sequencing metrics for libraries prepared from 100 ng of FFPE DNA for target capture, using either the KAPA HyperPrep Kit or Supplier I. Captures were performed with the SeqCap EZ Comprehensive Cancer Design (4 Mb) according to the manufacturer’s instructions, with the exception that the number of pre-capture amplification cycles for each library type was optimized based on post-ligation yields (10 cycles for KAPA HyperPrep Kit vs. 14 cycles for Supplier I libraries). All libraries were amplified for 13 cycles after capture. More efficient library construction and less PCR bias with the KAPA HyperPrep Kit resulted in higher library complexity prior to capture, lower duplication rates, and improved coverage. Sequencing (2 x 75 bp) was performed on an Illumina HiSeq 2500. Sequencing reads were down-sampled to ~14 million per library prior to analysis with Picard.

Improved performance with cell-free DNA

Ability to optimize adapter:insert ratios for challenging sample types, such as cell-free DNA
Higher library density compared to competitor kit*
Comparable duplication rates to vendor kits specific for low-input applications

See comparative data for improved library diversity using KAPA HyperPrep Kit compared to competitor kit. View data showing comparable duplication rates by KAPA HyperPrep Kit as those by a competitor kit designed specifically for low-input applications.

Figure 7. KAPA HyperPrep Kit enable improved sequencing metrics for libraries prepared from cell-free DNA. Library diversity and duplication rates for libraries prepared from 2 ng of cell-free DNA, using the KAPA HyperPrep Kit or a competitor low-input library construction kit (blue). Libraries were prepared according to manufacturer’s instructions. KAPA HyperPrep libraries were prepared with a range of adapter:insert molar ratios and the adaper:insert ratio of the competitor kit was unknown. Sequencing (2 x 150 bp) was performed on an Illumina MiSeq and data analyzed using Picard.

Figure 8. KAPA HyperPrep Kit enable improved sequencing metrics for libraries prepared from cell-free DNA. Library diversity and duplication rates for libraries prepared from 2 ng of cell-free DNA, using the KAPA HyperPrep Kit (green) or a competitor low-input library construction kit (red). Libraries were prepared according to manufacturer’s instructions. KAPA HyperPrep libraries were prepared with a range of adapter:insert molar ratios and the adaper:insert ratio of the competitor kit was unknown. Sequencing (2 x 150 bp) was performed on an Illumina MiSeq and data analyzed using Picard.

Data on file with Roche.

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All graphic data is on file, unless otherwise noted.

Specifications

Compatible Platform

All Illumina sequencing instruments
Library Type

DNA
Starting Material

Fragmented DNA, cDNA, PCR amplicons
Input Amount

1 ng – 1 µg

View Full Table

Cap colour	Component Name	Included in KAPA HyperPrep Kits (07962347001; 07962363001)	Included in KAPA HyperPrep Kits (PCR-free) (07962355001; 07962371001)
Purple	HyperPrep End Repair and A-Tailing Enzyme	Yes	Yes
Purple	HyperPrep End Repair and A-Tailing Buffer	Yes	Yes
Yellow	HyperPrep DNA Ligase Enzyme	Yes	Yes
Yellow	HyperPrep Ligation Buffer	Yes	Yes
Green	KAPA HiFi HotStart ReadyMix (2X)	Yes	No
Green	KAPA Library Amplification Primer Mix (10X)	Yes	No

Kits should be stored at -20˚C.

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Ordering

View Full Table

Kit Code	Roche Cat. No	Description	Kit Size (reactions)
KK8503	07962355001	KAPA HyperPrep Kit (PCR-free)	24
KK8505	07962371001	KAPA HyperPrep Kit (PCR-free)	96
KK8502	07962347001	KAPA HyperPrep Kit	24
KK8504	07962363001	KAPA HyperPrep Kit	96

Accessory Product

All KAPA Adapter Kits contain KAPA Adapter Dilution Buffer and three additional sealing films to support multiple use. KAPA Adapter Dilution Buffer (08278539001) is also available separately, if required.

View Full Table

Kit Code	Roche Cat. No	Description	Kit Size
KK8007	08963835001	KAPA HyperPure Beads (5 mL)	5 mL
KK8008	08963843001	KAPA HyperPure Beads (30 mL)	30 mL
KK8009	08963851001	KAPA HyperPure Beads (60 mL)	60 mL
KK8011	08963878001	KAPA HyperPure Beads (4 x 60 mL)	4 x 60 mL
KK8010	08963860001	KAPA HyperPure Beads (450 mL)	450 mL
KK8727	08861919702	KAPA Unique Dual-Indexed Adapters Kit (15 μM)	96 adapters x 20 μL each
KK8721	08278539001	KAPA Adapter Dilution Buffer (25 mL)	25 mL
N/A	09063781001	KAPA Universal Adapter, 15μM 960 μL	96-192* samples
N/A	09063790001	KAPA Universal Adapter, 15μM 960 μL	384-768* samples
N/A	09329862001	KAPA Universal UMI Adapter,960 μL	96-192* samples
N/A	09329889001	KAPA Universal UMI Adapter,4x960 μL	384 samples
N/A	09134336001	KAPA UDI Primer Mixes,1-96, 96 rxn	96 samples
N/A	09329838001	KAPA UDI Primer Mixes, 97-192, 96 rxn	96 samples
N/A	09329846001	KAPA UDI Primer Mixes,193-288, 96 rxn	96 samples
N/A	09329854001	KAPA UDI Primer Mixes, 289-384, 96 rxn	96 samples

*Workflow dependent

Additional pack sizes are available. Please speak to your local sales representative about how we can make it possible for you.

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What are the recommended applications for the KAPA HyperPrep Kit for Illumina platforms?

Whole-genome shotgun sequencing
Whole-exome or targeted sequencing, using KAPA HyperCap Target Enrichment Probes, Agilent SureSelect, IlluminaTruSeq, or IDT xGen Lockdown Probes, or other hybridization capture systems
Amplicon sequencing
ChIP-seq
Methyl-seq (in combination with the KAPA HiFi Uracil+ Library Amplification ReadyMix)

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 post-amplification cleanup.

Purified, adapter-ligated library DNA may be stored at 2°C to 8°C for 1 – 2 weeks, or at -15°C to -25°C for ≤1 month before amplification, target capture and/or sequencing. To avoid degradation, always store DNA in a buffered solution (10 mM Tris-HCl, pH 8.0 – 8.5) when possible, 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 mM EDTA
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 mM EDTA
Never shear DNA in water

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

KAPA Adapters are recommended for use with the KAPA HyperPrep Kits. However, the kit is also compatible with other full-length adapter designs wherein both the sequencing and cluster generation sequences are added during the ligation step, such as those routinely used in KAPA HyperCap and KAPA HyperPETE workflows, TruSeq (Illumina) and SureSelect XT2 (Agilent) kits, and other similar library construction 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.

For assistance with adapter compatibility and ordering, please contact Technical Support for guidelines on the formulation of user-supplied library amplification primers.

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 workflows 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.

KAPA Adapters may be used for all inputs with the appropriate dilution. For assistance with adapter compatibility and ordering, please visit sequencing.roche.com/support.

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

Where do I find more information about KAPA Adapters?

Please refer to the KAPA Adapter Technical Data Sheets for information about barcode sequences, pooling, kit configurations, formulation, and dilution for different KAPA DNA and RNA library preparation kits and inputs.

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 formation 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.

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.

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 support.

The estimated number of amplification cycles for libraries prepared from different amounts of input DNA can be found in the latest version of the KAPA HyperPrep Technical Data Sheet. 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.

Is library amplification required?

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 HyperPrep Kit enables PCR-free workflows with ≥ 50 ng input DNA. KAPA HyperPrep Kits without amplification reagents (07962355001 and 07962371001) are available for PCR-free workflows.

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

KAPA HyperPrep Kits contain a 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 us for guidelines on the formulation of user-supplied library amplification primers.

What is the shelf-life and the recommended storage conditions for KAPA HyperPrep Kits?

The enzymes provided in this kit are temperature sensitive, and appropriate care should be taken during shipping and storage. KAPA HyperPrep Kits are shipped on dry ice or ice packs, depending on the country of destination. Upon receipt, immediately store enzymes and reaction buffers at -15°C to -25°C in a constant-temperature freezer. When stored under these conditions and handled correctly, the kit components will retain full activity until the expiry date indicated on the kit label.

How many bead-based clean-ups are required after adapter-ligation?

The novel, one-tube KAPA HyperPrep 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. If necessary, a second post-ligation (or size selection step) cleanup may be included to remove all traces of unused adapter and adapter-dimer, especially for PCR-free workflows

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KAPA HyperPrep Kits