KAPA HyperPlus Kits

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

Overview

KAPA HyperPlus Kits provide a streamlined, automation-friendly workflow that includes fragmentation and library preparation in a single tube. This enzymatic fragmentation kit offers exceptional library construction efficiencies.

KAPA HyperPlus Kits offer a complete library preparation solution with KAPA Adapters and KAPA HyperPure Beads (sold separately). The kits are compatible with the Illumina sequencing platform.

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

Features and Benefits of KAPA HyperPlus Kits

Fully automatable DNA library construction in 1.5-2.5 hours with low-bias enzymatic fragmentation
Industry-leading conversion rates and library complexity,* particularly for FFPE DNA
Includes an additional End Repair and A-Tailing module for more sensitive applications with no change to the workflow
PCR-free workflows from lower inputs and improved sequence coverage in 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

*KAPA Adapter and KAPA Cleanup Beads Kits sold separately.

Figure 1 - The KAPA HyperPlus Workflow

Product highlights

Exceptional library construction yields

Low-input samples no longer require additional workflow modifications
PCR-free workflows possible from as little as 50 ng starting material
KAPA HyperPlus chemistry produce the highest post-ligation yields

View data for higher post-ligation yields produced by the KAPA HyperPlus Kit compared to Supplier N or Covaris-sheared DNA with KAPA HyperPrep Kit.

Figure 2. Post-ligation yields from a range of input amounts. The KAPA HyperPlus Kit (both original and upgraded workflows) converts more input DNA to adapter-ligated library than Supplier N or Covaris shearing combined with KAPA HyperPrep. Libraries were prepared from 1 ng, 10 ng and 100 ng E. coli gDNA using KAPA UDI Adapters and KAPA HyperPure Beads for all workflows, including Supplier N. Libraries were quantified after ligation using the KAPA Library Quantification Kit.

Tunable and low-bias enzymatic fragmentation

Library insert sizes adjustable from 150–800 bp by varying fragmentation time or temperature
Robust and reproducible fragmentation across a range of GC content and DNA input amounts and sample types

See performance data showing tunability and consistency in DNA library insert sizes with samples from different species, and comparative data showing minimal fragmentation bias by KAPA HyperPlus Kits compared to Supplier I as well as KAPA HyperPrep Kit utilizing Covaris-sheared DNA.

Figure 3. The KAPA fragmentation system enables tunable enzymatic fragmentation. Human genomic DNA (100 ng) was fragmented at 37°C for different periods of time (5 – 45 min) to achieve mode library insert sizes ranging from approximately 150 – 800 bp. The KAPA HyperPlus workflow was completed without any size selection using full-length adapters and 4 cycles of amplification. Final libraries were analyzed using a LabChip GX Touch HT instrument and HT DNA HiSens Reagent Kit (PerkinElmer).

Figure 4. Defined fragmentation parameters yield consistent library insert sizes across a range of species and genomic GC content. Genomic DNA (10 ng or 50 ng) from human (41% GC), Bordetella pertussis (68% GC), Clostridium difficile (29% GC), Escherichia coli (51% GC), and Plasmodium falciparum (20% GC) was fragmented at 37ºC for 5, 15, or 45 minutes, yielding average library insert sizes of ~700, ~350 or ~200 bp, respectively. Average fragment sizes for final, amplified libraries were determined with a Bioanalyzer 2100 instrument and High-Sensitivity DNA Assay (Agilent Technologies).

Figure 5. The KAPA HyperPlus workflow results in minimal fragmentation bias. Nucleotide content over a 40 bp window (-10 to +30 bp relative to read alignment start) for whole exome libraries were prepared from 50 ng of human gDNA (41% GC) with the KAPA HyperPlus Kit, the KAPA HyperPrep Kit utilizing Covaris-sheared DNA, or Supplier I. Enzymatic fragmentation with the KAPA HyperPlus Kit resulted in slightly more start site bias than mechanical shearing, but much less bias than tagmentation. Paired-end sequencing (2 x 100 bp) was performed on an Illumina HiSeq 2500 and analysis performed with a custom python script.

Figure 6. The KAPA HyperPlus workflow results in minimal fragmentation bias. Nucleotide content over a 40 bp window (-10 to +30 bp relative to read alignment start) for whole exome libraries were prepared from 50 ng of human gDNA (41% GC) with the KAPA HyperPlus Kit, the KAPA HyperPrep Kit utilizing Covaris-sheared DNA, or Supplier I. Enzymatic fragmentation with the KAPA HyperPlus Kit resulted in slightly more start site bias than mechanical shearing, but much less bias than tagmentation. Paired-end sequencing (2 x 100 bp) was performed on an Illumina HiSeq 2500 and analysis performed with a custom python script.

Figure 7. The KAPA HyperPlus workflow results in minimal fragmentation bias. Nucleotide content over a 40 bp window (-10 to +30 bp relative to read alignment start) for whole exome libraries were prepared from 50 ng of human gDNA (41% GC) with the KAPA HyperPlus Kit, the KAPA HyperPrep Kit utilizing Covaris-sheared DNA, or Supplier I. Enzymatic fragmentation with the KAPA HyperPlus Kit resulted in slightly more start site bias than mechanical shearing, but much less bias than tagmentation. Paired-end sequencing (2 x 100 bp) was performed on an Illumina HiSeq 2500 and analysis performed with a custom python script.

Superior sequencing results with KAPA HyperPlus Kits

Higher post-ligation yields result in fewer amplification cycles and lower duplication rates
Detect low-frequency mutations with high confidence due to decreased sequencing artifacts and increased sensitivity with the upgraded version of the KAPA HyperPlus Kit

View comparative data showing similar duplication rates and sensitivity, with improved sequencing results using the upgraded KAPA HyperPlus Kit compared to KAPA HyperPrep Kits utilizing Covaris-sheared DNA.

Figure 8. Lower duplication rates. Whole human exome libraries were prepared using 100 ng inputs with the original and upgraded KAPA HyperPlus Kit. Captures were performed with the SeqCap EZ MedExome panel.

Figure 9. Improved sequencing performance. Whole human exome libraries were prepared using 100 ng inputs with the original and upgraded KAPA HyperPlus Kit. Captures were performed with the SeqCap EZ MedExome panel. Strand-split artifact reads (SSARs) represent chimeric reads that appear to be derived from non-contiguous portions of the genome¹.

¹ Source: Haile, et al. (2019) Sources of erroneous sequences and artifact chimeric reads in next generation sequencing of genomic DNA from formalin-fixed paraffin-embedded samples. Nucleic Acids Research, 2019, 47,2.

Figure 10. Higher single nucleotide polymorphism (SNP) sensitivity. Whole human exome libraries were prepared using 100 ng high-quality gDNA with the KAPA HyperPrep Kit, the original and upgraded KAPA HyperPlus Kit. Captures were performed with the SeqCap EZ MedExome panel.

Improved coverage depth for FFPE samples compared to Covaris-sheared DNA

Significant increase in coverage depth compared to KAPA HyperPrep Kits using Covaris-sheared DNA
Lower duplication rates, more on-target reads and more bases covered compared to KAPA HyperPrep Kits using Covaris-sheared DNA
Detection of variants with high degree of correlation between libraries prepared from KAPA HyperPrep Kits using Covaris-sheared DNA versus KAPA HyperPlus Kits*

View performance data showing significantly higher coverage for FFPE samples using KAPA HyperPlus Kits compared to Covaris-sheared DNA.

Figure 11. KAPA HyperPlus Kit enable improved coverage for FFPE samples. Libraries were prepared from 200 ng of FFPE DNA or 50 ng of matched normal controls (DNA from blood) using the KAPA HyperPrep Kit utilizing Covaris-sheared DNA, or the KAPA HyperPlus Kit. Two rounds of multiplexed target capture were performed with a custom SeqCap EZ panel targeting regions of 42 genes associated with gastrointestinal cancer. Significantly lower duplication rates for the KAPA HyperPlus libraries and slightly higher on-target reads translated to a significant increase in coverage depth for the FFPE samples. Variants at an expected frequency ≥5% were detected with a high degree of correlation (R2 >0.96) between the libraries prepared from Covaris-sheared vs. enzymatically fragmented DNA. Paired-end sequencing (2 x 150 bp) was performed on an Illumina MiSeq and data analyzed with Picard and Illumina VariantStudio v2.2. Data courtesy of The Royal Marsden Hospital.

Figure 12. KAPA HyperPlus Kit enable improved coverage for FFPE samples. Libraries were prepared from 200 ng of FFPE DNA or 50 ng of matched normal controls (DNA from blood) using the KAPA HyperPrep Kit utilizing Covaris-sheared DNA, or the KAPA HyperPlus Kit. Two rounds of multiplexed target capture were performed with a custom SeqCap EZ panel targeting regions of 42 genes associated with gastrointestinal cancer. Significantly lower duplication rates for the KAPA HyperPlus libraries and slightly higher on-target reads translated to a significant increase in coverage depth for the FFPE samples. Variants at an expected frequency ≥5% were detected with a high degree of correlation (R2 >0.96) between the libraries prepared from Covaris-sheared vs. enzymatically fragmented DNA. Paired-end sequencing (2 x 150 bp) was performed on an Illumina MiSeq and data analyzed with Picard and Illumina VariantStudio v2.2. Data courtesy of The Royal Marsden Hospital.

Figure 13. KAPA HyperPlus Kit enable improved coverage for FFPE samples. Libraries were prepared from 200 ng of FFPE DNA or 50 ng of matched normal controls (DNA from blood) using the KAPA HyperPrep Kit utilizing Covaris-sheared DNA, or the KAPA HyperPlus Kit. Two rounds of multiplexed target capture were performed with a custom SeqCap EZ panel targeting regions of 42 genes associated with gastrointestinal cancer. Significantly lower duplication rates for the KAPA HyperPlus libraries and slightly higher on-target reads translated to a significant increase in coverage depth for the FFPE samples. Variants at an expected frequency ≥5% were detected with a high degree of correlation (R2 >0.96) between the libraries prepared from Covaris-sheared vs. enzymatically fragmented DNA. Paired-end sequencing (2 x 150 bp) was performed on an Illumina MiSeq and data analyzed with Picard and Illumina VariantStudio v2.2. Data courtesy of The Royal Marsden Hospital.

Figure 14. KAPA HyperPlus Kit enable improved coverage for FFPE samples. Libraries were prepared from 200 ng of FFPE DNA or 50 ng of matched normal controls (DNA from blood) using the KAPA HyperPrep Kit utilizing Covaris-sheared DNA, or the KAPA HyperPlus Kit. Two rounds of multiplexed target capture were performed with a custom SeqCap EZ panel targeting regions of 42 genes associated with gastrointestinal cancer. Significantly lower duplication rates for the KAPA HyperPlus libraries and slightly higher on-target rates translated to a significant increase in coverage depth for the FFPE samples. Variants at an expected frequency ≥5% were detected with a high degree of correlation (R2 >0.96) between the libraries prepared from Covaris-sheared vs. enzymatically fragmented DNA. Paired-end sequencing (2 x 150 bp) was performed on an Illumina MiSeq and data analyzed with Picard and Illumina VariantStudio v2.2. Data courtesy of The Royal Marsden Hospital.

Minimal sequence coverage bias

Lower sequence bias when compared to other enzymatic fragmentation methods
Less bias leads to more uniform sequencing coverage and reduced sequencing costs

View GC-bias plots showing superior performance using KAPA HyperPlus Kits compared to Supplier N and Supplier Q workflows.

Figure 15. GC bias comparision. GC bias for C. difficile, E. coli and B. pertussis was assessed by calculating the GC content of the reference in 100 bp and bins and plotting normalized coverage across these bins for the KAPA HyperPlus, Supplier N and Supplier Q workflows, using Picard CollectGCBiasMetrics. Libraries were prepared from 10 ng of input DNA. In the absence of sequencing bias, all bins would be equally represented, indicated by a horizontal distribution centered on a normalized coverage of 1. Distribution of GC content in the genome is indicated by the grey histograms.

Figure 16. GC bias comparision. GC bias for C. difficile, E. coli and B. pertussis was assessed by calculating the GC content of the reference in 100 bp and bins and plotting normalized coverage across these bins for the KAPA HyperPlus, Supplier N and Supplier Q workflows, using Picard CollectGCBiasMetrics. Libraries were prepared from 10 ng of input DNA. In the absence of sequencing bias, all bins would be equally represented, indicated by a horizontal distribution centered on a normalized coverage of 1. Distribution of GC content in the genome is indicated by the grey histograms.

Figure 17. GC bias comparision. GC bias for C. difficile, E. coli and B. pertussis was assessed by calculating the GC content of the reference in 100 bp and bins and plotting normalized coverage across these bins for the KAPA HyperPlus, Supplier N and QIAseq FX workflows, using Picard CollectGCBiasMetrics. Libraries were prepared from 10 ng of input DNA. In the absence of sequencing bias, all bins would be equally represented, indicated by a horizontal distribution centered on a normalized coverage of 1. Distribution of GC content in the genome is indicated by the grey histograms.

*Data on file with Roche.

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Specifications

Compatible Platform

All Illumina sequencing instruments
Library Type

DNA
Starting Material

genomic DNA, FFPE DNA, amplicons
Input Amount

1 ng – 1 µg
Kit storage

-15°C to -25°C

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Cap colour	Component Name	Included in KAPA HyperPlus Kits (07962401001; 07962428001)	Included in KAPA HyperPlus Kits (PCR-free) (07962410001; 07962436001)
Red	KAPA Frag Enzyme	Yes	Yes
Blue	KAPA Frag Buffer (10X)	Yes	Yes
White	Conditioning Solution	Yes	Yes
Orange	HyperPlus End Repair and A-Tailing Enzyme	Yes	Yes
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

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Ordering

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Kit Code	Roche Cat. No	Description	Kit Size
KK8512	07962401001	KAPA HyperPlus Kit	24
KK8514	07962428001	KAPA HyperPlus Kit	96
KK8513	07962410001	KAPA HyperPlus Kit (PCR-free)	24
KK8515	07962436001	KAPA HyperPlus Kit (PCR-free)	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.

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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)	25mL
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 for KAPA HyperPure Beads. Please speak to your local sales representative.

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What are the recommended applications for the KAPA HyperPlus Kit?

Whole-genome shotgun sequencing
Whole-exome or targeted sequencing, using KAPA Target Enrichment Probes, Agilent SureSelect, IlluminaTruSeq, or IDT xGen^® Lockdown^® Probes, or other hybridization capture systems

Are there safe stopping points in the library construction process?

The library construction process, from enzymatic fragmentation to final library, can be performed in 1.5 to 2.5 hours depending on experience, the number of samples being processed, and whether or not library amplification is performed. If necessary, the protocol may be paused safely 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-HCI, pH 8.0 - 8.5) when possible, and minimize the number of freeze-thaw cycles.

How do I select which End Repair & A-Tailinq Enzyme Mix to use?

Two workflows are available for the End Repair and A-tailing step. This protocol has been validated for use with either the HyperPrep End Repair & A-Tailing Enzyme Mix (existing chemistry) or the HyperPlus End Repair & A-Tailing Enzyme Mix (enhanced chemistry). The formulation of the HyperPlus End Repair & A-Tailing Enzyme Mix has been enhanced to improve performance for more sensitive assays. There are no changes to the KAPA HyperPlus workflow, except the option exists to use either of the End Repair & A-Tailing Enzyme Mixes during the End Repair and A-tailing step.

For all new applications, it is recommended to use the HyperPlus End Repair & A-Tailing Enzyme Mix (enhanced chemistry). For existing validated workflows, it is recommended to perform a side-by-side comparison using the two enzyme mixes and determine which one is most suitable for the specific

Are there specific fragmentation recommendations for FFPE samples?

DNA quality impacts the fragmentation of FFPE DNA. The guidelines presented in the Technical Data Sheet are a good starting point for FFPE samples with a Q129/Q41 ratio of 0.4 or higher. However, longer fragmentation times may improve results for lower-quality FFPE samples.

Longer fragmentation times typically increase the proportion of input DNA converted to fragments in the 150 - 250 bp range, reduce residual high-molecular weight DNA, and correlate with higher yields during library construction.

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

KAPA Adapters are recommended for use with the KAPA HyperPlus 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 Target Enrichment probes, 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 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 Adapters 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 testinq 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.

How manv bead-based clean-ups are required after adapter-liqation?

The novel, one-tube KAPA HyperPlus 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.

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 theoretical number of cycles required to obtain approximately 1 μg of amplified library DNA from adapter-ligated library DNA, shown in the latest version of the KAPA HyperPlus Technical Data Sheet.

Is library amplification required?

Depending on the amount of library material required for your application, it may be possible to omit library amplification. In such cases, it is important to ensure that your adapters are designed to support sample indexing (where required), cluster amplification and sequencing. Omitting library amplification further streamlines the workflow and reduces overall library preparation time to ≤ 1.5 hours. The high conversion efficiency achievable with the KAPA HyperPlus Kit enables PCR-free workflows from as little as 50 ng of input DNA. KAPA HyperPlus Kits without amplification reagents (07962410001 and 07962436001) are available for PCR-free workflows.

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) can provide useful data for protocol optimization and troubleshooting.

What is the shelf-life and the recommended storaqe conditions for KAPA HvperPlus Kits?

The enzymes provided in this kit are temperature sensitive, and appropriate care should be taken during shipping and storage KAPA HyperPlus 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.

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KAPA HyperPlus Kits