Technical Data Sheet

KAPA HyperPlus Kit Kapa/Roche Kit Codes and Components

KAPA Frag Enzyme 100 µL
KR1145 – v4.17 KAPA Frag Buffer (10X) 50 µL
KK8510 Conditioning Solution 580 µL
07962380001
End Repair & A-Tailing Buffer 70 µL
This Technical Data Sheet provides product information
KK8511* End Repair & A-Tailing Enzyme 30 µL
and a detailed protocol for the KAPA HyperPlus Kit. 07962398001 Ligation Buffer 300 µL
This document applies to KAPA HyperPlus Kits 8 libraries DNA Ligase 100 µL
(07962380001, 07962401001 and 07962428001), KAPA HiFi HotStart ReadyMix (2X)* 250 µL
Library Amplification Primer Mix (10X)* 50 µL
and KAPA HyperPlus Kits for PCR-free workflows
(07962398001, 07962410001 and 07962436001). KAPA Frag Enzyme 270 µL
KAPA Frag Buffer (10X) 140 µL
KK8512 Conditioning Solution 580 µL
Contents 07962401001
End Repair & A-Tailing Buffer 210 µL
KK8513* End Repair & A-Tailing Enzyme 90 µL
Product Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 07962410001 Ligation Buffer 900 µL
Product Applications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 24 libraries DNA Ligase 300 µL
KAPA HiFi HotStart ReadyMix (2X)* 690 µL
Product Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Library Amplification Primer Mix (10X)* 138 µL
Shipping and Storage . . . . . . . . . . . . . . . . . . . . . . . . . 2 KAPA Frag Enzyme 1.27 mL
Handling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 KAPA Frag Buffer (10X) 640 µL
KK8514 Conditioning Solution 580 µL
Quality Control. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 07962428001
End Repair & A-Tailing Buffer 930 µL
KK8515* End Repair & A-Tailing Enzyme 400 µL
Important Parameters. . . . . . . . . . . . . . . . . . . . . . . . . . . 3 07962436001 Ligation Buffer 3.8 mL
Automated Library Construction . . . . . . . . . . . . . . . . . 3 96 libraries DNA Ligase 1.26 mL
Safe Stopping Points. . . . . . . . . . . . . . . . . . . . . . . . . . 3 KAPA HiFi HotStart ReadyMix (2X)* 3.0 mL
Library Amplification Primer Mix (10X)* 600 µL
Input DNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
*KK8511, KK8513, and KK8515 are available for PCR-free workflows, and do not
Fragmentation Parameters. . . . . . . . . . . . . . . . . . . . . . 5 contain library amplification reagents.

Adapter Design and Concentration. . . . . . . . . . . . . . . 5

Quick Notes
Post-ligation Processing . . . . . . . . . . . . . . . . . . . . . . . 6
Reaction Cleanups. . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 • This kit provides a versatile, streamlined DNA
fragmentation and library construction protocol.
Size Selection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Libraries for Illumina sequencing may be prepared
Library Amplification. . . . . . . . . . . . . . . . . . . . . . . . . . 7 from a wide range of DNA samples and inputs
Evaluating the Success of Library Construction . . . . . 8 (1 ng – 1 µg) in 1.5 – 3 hrs.
Process Workflow. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 • The novel one-tube DNA fragmentation and library
construction chemistry improves library yield and
Library Construction Protocol. . . . . . . . . . . . . . . . . . . 12 quality, particularly for FFPE and low-input libraries.
Appendix 1: Size Selection. . . . . . . . . . . . . . . . . . . . . . . 15 • The protocol is easy to automate. Generous
reagent excesses are supplied in 96-reaction kits to
Appendix 2: Optimization of Fragmentation
accommodate automated liquid handling.
Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
• This kit contains all the reagents for DNA
Appendix 3: Library Construction Guidelines fragmentation, library construction, and high
for the Roche SeqCap EZ Target Capture System. . . . . 18 efficiency and low bias library amplification except
Restrictions and Liabilities. . . . . . . . . . . . . . . . . . . . . . . 20 for adapters and beads. KAPA Pure Beads and
KAPA Adapters are sold separately. Kits without
Note to Purchaser: Limited Product Warranty . . . . . . . . 20 an amplification module are available for PCR-free
Note to Purchaser: Limited License . . . . . . . . . . . . . . . . 20 workflows.
• The Process Workflow provides an overview of
the DNA fragmentation and library construction
process. Appendix 1 provides a size selection
protocol. Appendix 2 contains guidelines for
optimization of fragmentation. Appendix 3 provides
details for integration with the SeqCap EZ workflow.
• If your input DNA contains EDTA, please consult the
Important Parameters: Input DNA and additional
guidelines in Appendix 2.

Effective date: September 2017 For Research Use Only. Not for use in diagnostic procedures.
KAPA HyperPlus Kit Technical Data Sheet
Product Description Product Applications
The KAPA HyperPlus Kit provides a versatile, streamlined KAPA HyperPlus Kits are ideally suited for low- and
DNA fragmentation and library construction protocol for high-throughput NGS library construction workflows
the rapid preparation of libraries for Illumina sequencing. that require DNA fragmentation, end repair, A-tailing,
The novel one-tube chemistry and protocol improves adapter ligation and library amplification (optional).
the efficiency and consistency of library construction, Kits are designed for library construction from a wide range
and yields libraries of similar or better quality than those of sample types and inputs (1 ng – 1 µg), and are compatible
produced with the KAPA Hyper Prep Kit from Covaris- with complex, genomic DNA; low-complexity samples such
sheared DNA. It outperforms tagmentation-based as small viral genomes, plasmids, cDNA and long amplicons;
workflows in terms of robustness, flexibility and sequence and low-quality DNA such as FFPE samples.
coverage and uniformity.
The entire workflow is automation-friendly and may be
The workflow combines enzymatic steps and employs incorporated into workflows for a wide range of NGS
minimal bead-based cleanups, thereby reducing sample applications, including:
handling and overall library preparation time to 1.5 – 3 hrs.
• whole-genome, shotgun sequencing
The kit contains all of the enzymes and reaction buffers
required for: • whole exome or targeted sequencing, using Roche
SeqCap EZ, Agilent SureSelect, Illumina TruSeq, or IDT
1. enzymatic fragmentation to produce dsDNA fragments;
xGen Lockdown Probes, or other hybridization capture
2. end repair and A-tailing to produce end-repaired, systems
5'-phosphorylated, 3'-dA-tailed dsDNA fragments;
• RNA-seq (starting with cDNA).
3. adapter ligation, during which dsDNA adapters
with 3'-dTMP overhangs are ligated to 3'-dA-tailed
molecules; and Product Specifications
4. library amplification (optional), which employs high- Shipping and Storage
fidelity, low-bias PCR to amplify library fragments The enzymes provided in this kit are temperature sensitive,
carrying appropriate adapter sequences on both ends. and appropriate care should be taken during shipping and
storage. KAPA HyperPlus Kits are shipped on dry ice.
The kit provides a single concentrated buffer and a single Upon receipt, immediately store enzymes and reaction
enzyme mixture for enzymatic fragmentation, as well as buffers at -15°C to -25°C in a constant-temperature
for each of the two library construction steps—but does freezer. When stored under these conditions and handled
not include adapters or beads required for cleanups after correctly, the kit components will retain full activity until
adapter ligation and library amplification. KAPA Pure the expiry date indicated on the kit label.
Beads and KAPA Adapters are sold separately. Handling
In order to maximize sequence coverage uniformity, Always ensure that KAPA HyperPlus Kit components
it is critical to minimize library amplification bias. KAPA have been fully thawed and thoroughly mixed before use.
HiFi DNA Polymerase is designed for low-bias, high- The End Repair & A-Tailing Buffer and Ligation Buffer may
fidelity PCR, and is the reagent of choice for NGS library contain precipitates when thawed at 2°C to 8°C. These
amplification.1,2,3,4 KAPA HyperPlus Kits include KAPA buffers must be thawed at room temperature and vortexed
HiFi HotStart ReadyMix (2X), a ready-to-use PCR mix thoroughly before use. KAPA HiFi HotStart ReadyMix (2X)
comprising all the components for library amplification— contains isostabilizers and may not freeze completely,
except primers and template. Kits also include Library even when stored at -15°C to -25°C. Nevertheless, always
Amplification Primer Mix (10X), designed for the high- ensure that the ReadyMix is fully thawed and thoroughly
efficiency amplification of Illumina libraries flanked by mixed before use. Reaction master mixes prepared from
adapters containing the P5 and P7 flow cell sequences. the enzymes and buffers for fragmentation, end repair
Kits without the amplification module (KK8511, and A-tailing, as well as for ligation, are very viscous and
KK8513, KK8515) are available for PCR-free workflows. require special attention during pipetting. Keep all enzyme
They may also be combined with KAPA HiFi Real-time components and master mixes on ice as long as possible
Library Amplification Kits (KK2701, KK2702) for more during handling and preparation.
precise control over library amplification.
Quality Control
1. Oyola, S.O., et al., BMC Genomics 13, 1 (2012).
2. Quail, M.A., et al., Nature Methods 9, 10 (2012). All kit components are subjected to stringent functional
3. Quail, M.A., et al., BMC Genomics 13, 341 (2012). quality control, are free of detectable contaminating exo-
4. Ross, M.G., et al., Genome Biology 14, R51 (2013). and endonuclease activities, and meet strict requirements
with respect to DNA contamination. Please contact
Technical Support at [Link]/support for
more information.

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

KAPA HyperPlus Kit Technical Data Sheet
Important Parameters long periods of time, particulary if they are not actively
cooled. Prepare library amplification master mixes
Library construction workflows must be tailored and freshly before use, or dispense primer mixes separately
optimized to accommodate specific experimental from the KAPA HiFi HotStart ReadyMix.
designs, sample characteristics, sequencing applications
and equipment. The protocol provided in this document • Since an excess (5 – 20%) of each reagent master
is generic, and reaction parameters may be adjusted mix will be required, generous reagent overages are
as required to optimize performance, efficiency and included in 96-reaction kits. The appropriate excess
cost-effectiveness. for other reagents (adapters, beads, 80% ethanol and
elution buffer) varies from one liquid handling system
In addition to the information in this section, please to another.
consult the KAPA NGS Library Preparation
Technical Guide and/or contact Technical Support at • Enzymatic fragmentation reactions must be set up in
[Link]/support for further guidelines a plate that is actively cooled to 4°C, and moved to
when designing or optimizing your library construction a second, pre-equilibrated Peltier device or on-deck
workflow. thermocycler for the fragmentation incubation (typically
performed at 37°C). After fragmentation, immediately
Automated Library Construction return the plate to 4°C, add end repair and A-tailing
The KAPA HyperPlus workflow described in this document reagents at 4°C, and move the plate to an on- or off-
is designed to be “automation friendly” and can be deck cycler for the 65°C incubation without delay.
performed manually, or in a semi- or fully automated • Incubations at temperatures above 50°C must be
fashion using a suitable automated liquid handling performed in a thermocycler with a heated lid.
platform. In addition to increased sample throughput,
automation may be expected to provide additional • This protocol has been optimized for 96-well PCR
advantages such as improved reproducibility and process plates with a maximum working volume of ~200 µL.
control. However, automation may result in slightly lower Plates with larger working volumes or deep-well plates
yields and/or different size distributions when compared may be used to accommodate larger reaction volumes
with manual library construction performed by a skilled, if needed.
experienced and attentive technician. Most often, these • Always use plastics that are certified to be nuclease-
discrepancies can be minimized through careful selection free. Low DNA-binding plastics are recommended.
of hardware and plasticware, and optimization of liquid When selecting the most appropriate plasticware for
handling parameters. your workflow, consider compatibility with:
Kapa Biosystems does not supply automated liquid -- the plate gripper and other components of your
handling equipment, but collaborates with automation liquid handling system;
solution providers and customers to develop and
qualify optimized automated methods for our kits, for -- the magnet used during bead manipulations;
liquid handling platforms routinely used in NGS library -- Peltier devices or thermocyclers used for reaction
construction. Please contact your instrument vendor or incubations and/or library amplification.
visit [Link]/support if you are interested
in using the KAPA HyperPlus Kit with your particular • Design automated methods in a manner that ensures
automated liquid handling system. the highest consistency across all 12 columns of
96-well working plates, and eliminates all possible
When attempting to develop an automated KAPA sources of sample-to-sample and environmental
HyperPlus method, please keep the following in mind: contamination. Consider performing pre- and post-
• Reaction components for enzymatic reactions should PCR steps on dedicated instruments, if available.
be combined into master mixes, rather than dispensed
separately. The enzymatic fragmentation master mix Safe Stopping Points
must be made fresh, and actively cooled at 4°C. In The library construction process, from enzymatic
contrast, the master mixes for end repair and A-tailing, fragmentation to final library, can be performed in
as well as for adapter ligation, are stable for ≤24 hrs 1.5 to 3 hrs—depending on experience, the number of
at room temperature, and do not have to be actively samples being processed, and whether or not library
cooled during automated library construction. amplification is performed. If necessary, the protocol may
be paused safely after completion of the Post-ligation
• Master mixes for fragmentation, the end repair and
Cleanup (step 4.17; the end of the protocol for PCR-free
A-tailing reaction, as well as for adapter ligation, are
workflows).
highly viscous, and require careful optimization of
pipetting parameters. 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
• Due to the strong 3'g5' exonuclease activity of KAPA
≤1 month before amplification, target capture and/or
HiFi HotStart DNA Polymerase, PCR master mixes
sequencing. Library amplification products may be stored
with primers should preferably not be left on-deck for

For Research Use Only. Not for use in diagnostic procedures. 3

KAPA HyperPlus Kit Technical Data Sheet
in a similar way, but the post-amplification cleanup should based cleanup with KAPA Pure Beads. Please refer to the
be performed as soon as possible. To avoid degradation, KAPA Pure Beads Technical Data Sheet for a detailed
always store DNA in a buffered solution (10 mM Tris-HCl, DNA cleanup protocol. For optimal fragmentation
pH 8.0 – 8.5) when possible, and minimize the number of results, elute DNA in 10 mM Tris-HCl (pH 8.0 - 8.5) after
freeze-thaw cycles. the cleanup.
Input DNA • DNA isolated from blood samples has been reported
• This protocol is suitable for fragmentation and library to contain inhibitors, which can affect the efficiency of
construction from 1 ng – 1 µg of double-stranded fragmentation. Performing a 3X bead-based cleanup
DNA. Please refer to Table 1 for recommended inputs prior to fragmentation is recommended.
of different types of DNA, for different sequencing • Bead-based cleanups to remove EDTA from FFPE DNA
applications. samples may not yield comparable results. Recovery of
FFPE DNA may be low, and not always proportional to
Table 1. Recommended inputs into library construction
DNA quality. For FFPE DNA, neutralization of EDTA with
Recommended the Conditioning Solution (see below) is recommended
Application Sample type
input as a first approach.
Complex gDNA
WGS
(high quality)
50 ng – 1 µg • If a DNA cleanup is not feasible, the inhibitory effect
of the EDTA can be mitigated by the inclusion
Target capture
Complex gDNA of Conditioning Solution at the appropriate final
(WES, custom 10 ng – 1 µg
(high quality) concentration in the fragmentation reaction.
panels)
WGS, target
FFPE DNA
≥50 ng • To facilitate reaction setup, the Conditioning Solution
capture (quality dependent) is pre-diluted to the appropriate working concentration
WGS Microbial DNA 1 ng – 1 µg as outlined in Table 3, and a fixed volume (5 µL) is
≥50 ng (no SS)* included in the fragmentation reaction. Please note
WGS (PCR-free) High-quality DNA that dilution of the Conditioning Solution is based on
≥200 ng (w/SS)*
Targeted the final concentration of EDTA in the fragmentation
Long amplicons ≥1 ng reaction (once input DNA has been diluted in a volume
sequencing
Full-length/ of 50 µL), and not on the EDTA concentration in the
RNA-seq unfragmented ≥1 ng DNA preparation.
cDNA
*SS = size selection; results in the loss of 60 – 95% of DNA, irrespective of whether
Table 3. Conditioning Solution dilutions for DNA samples
a bead- or gel-based technique is used. containing EDTA
Volume of Volume of
• The proportion of fragmented DNA that is successfully Final EDTA
Dilution conditioning PCR-grade
concentration
converted to adapter-ligated molecules decreases as in 50 µL rxn
factor solution water
input is reduced. Table 2 summarizes the expected (per 100 µL) (per 100 µL)
conversion rates for different DNA input ranges. These 0.02 – 0.05 mM 32.0 3.1 µL 96.9 µL
figures apply to high-quality DNA, and may be lower for 0.1 mM 15.4 6.5 µL 93.5 µL
DNA of lower quality, e.g., FFPE samples. Workflows
0.2 mM 7.4 13.5 µL 86.5 µL
with additional cleanups or size selection prior to
adapter ligation are also likely to result in a lower yield 0.3 mM 4.8 21.0 µL 79.0 µL
of adapter-ligated molecules. 0.4 mM 3.3 30.0 µL 70.0 µL
0.5 mM 2.6 38.8 µL 61.2 µL
Table 2. Expected conversion rates for DNA input ranges
0.6 mM 2.2 46.5 µL 53.5 µL
DNA input Expected conversion rate
0.7 mM 1.8 56.0 µL 44.0 µL
1 – 10 ng 5 – 20%
0.8 mM 1.6 64.0 µL 36.0 µL
11 – 100 ng 10 – 50%
0.9 mM 1.4 72.0 µL 28.0 µL
>100 ng 50 – 100%
1.0 mM 1.3 80.0 µL 20.0 µL

• The enzymatic fragmentation reaction is very

• Prepare a minimum of 100 µL of diluted Conditioning
sensitive to the presence of EDTA, which must be
Solution (as indicated in Table 3), or calculate the
removed or neutralized prior to fragmentation. EDTA
volume needed using the following formula:
in DNA preparations is usually introduced via elution
buffers used in the final stages of the DNA extraction (number of reactions x 5 µL) + 10% excess
or purification process. • The addition of Conditioning Solution to fragmentation
• Removal of EDTA from DNA samples prior to reactions will lead to suboptimal results if your DNA
fragmentation is recommended to ensure consistent does not contain EDTA, or if the final concentration
results. This may be achieved by means of a 3X bead- of the Conditioning Solution is not matched to the

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

KAPA HyperPlus Kit Technical Data Sheet
final EDTA concentration in the reaction. identical fragmentation profiles for the same sample
and input, and fragmentation times may have to be
• Please refer to Appendix 2: Optimization of
modified slightly when switching between devices.
Fragmentation Parameters (p. 16) for more guidelines
The relative impact of the device used for the
regarding the processing of EDTA-containing
fragmentation incubation is likely to be less significant
DNA samples.
as fragmentation time increases.
Fragmentation Parameters
Adapter Design and Concentration
• The fragmentation parameters provided in the Library
• KAPA Adapters are recommended for use with the
Construction Protocol (step 1, p. 12) apply to the
KAPA HyperPlus Kit. However, the kit is also compatible
fragmentation of high-quality genomic DNA.
with other full-length adapter designs wherein both
• The degree of fragmentation (mode size and size the sequencing and cluster generation sequences are
distribution of DNA fragments) is controlled by added during the ligation step, such as those routinely
fragmentation time and temperature, and both factors used in TruSeq (Illumina), SeqCap EZ (Roche) and
may be modulated to achieve the desired results. SureSelect XT2 (Agilent) kits, and other similar library
construction workflows. Custom adapters that are of
• DNA quality impacts the fragmentation of FFPE DNA.
similar design and are compatible with “TA-ligation” of
The guidelines in the Library Construction Protocol
dsDNA may also be used, remembering that custom
(step 1, p. 12) are a good starting point for FFPE
adapter designs may impact library construction
samples with a Q129/Q41 ratio of ~0.4 or higher (as
efficiency. For assistance with adapter compatibility
determined with the KAPA hgDNA Quantification and
and ordering, please contact Technical Support at
QC Kit). However, slightly longer fragmentation times
[Link]/support.
may improve results for lower-quality FFPE samples.
Longer fragmentation times typically increase the • Adapter concentration affects ligation efficiency,
proportion of input DNA converted to fragments in the as well as adapter and adapter-dimer carry-over
150 – 250 bp range, reduce residual high-molecular during the post-ligation cleanup. The optimal
weight DNA, and correlate with higher yields during adapter concentration for your workflow represents a
library construction. compromise between the above factors and cost.
• Standard fragmentation parameters may result in over- • Ligation efficiency is robust for adapter:insert molar
fragmentation of low-complexity samples, such as ratios ranging from 10:1 to >200:1, making it unnec-
small viral genomes, plasmids, long amplicons and essary to adjust adapter stock concentrations to
cDNA. For these sample types, the fragmentation time accommmodate moderate variations in DNA input or
may have to be reduced to 5 min or less to achieve the fragment length. Please refer to Table 4 for the recom-
desired mode fragment length. This makes control over mended adapter concentrations for different DNA inputs.
the reaction difficult, particularly when a large number
• High adapter:insert molar ratios (>200:1) are beneficial
of samples are processed manually. To enable more
for low-input applications. When optimizing workflows
robust and reproducible results, the fragmentation
for DNA inputs ≤25 ng, two or three adapter concen-
temperature may be decreased (to 30°C or 25°C) to
trations should be evaluated: try the recommended
reduce enzymatic activity, thus increasing the time
adapter concentration (Table 4), as well as one or two
needed to achieve the desired fragment length.
additional concentrations in a range that is 2 – 10 times
• Please refer to Appendix 2 (p. 16) for guidelines on how higher than the recommended concentration.
to systematically optimize fragmentation parameters
• Adapter quality has an impact on the effective
for your specific samples.
concentration of adapter available for ligation. Always
• Different devices (e.g., a thermocycler vs. heating source the highest quality adapters from a reliable
block, or different Peltier devices integrated into supplier, dilute and store adapters in a buffered solution
automated liquid handling systems) may not yield with the requisite ionic strength, and avoid excessive

Table 4. Recommended adapter concentrations for libraries constructed from 1 ng – 1 µg input DNA*
Adapter stock Adapter:insert Adapter stock Adapter:insert
Input DNA Input DNA
concentration molar ratio concentration molar ratio
1 µg 15 µM 10:1 25 ng 7.5 µM 200:1
500 ng 15 µM 20:1 10 ng 3 µM 200:1
250 ng 15 µM 40:1 5 ng 1.5 µM 200:1
100 ng 15 µM 100:1 2.5 ng 750 nM 200:1
50 ng 15 µM 200:1 1 ng 300 nM 200:1
*Adapter:insert molar ratio calculations are based on a mode DNA fragment length of 200 bp, and will be higher for longer DNA fragments, or slightly lower for DNA fragmented
to a mode size <200 bp. The lower adapter:insert molar ratios recommended for inputs >100 ng represent a fair compromise between library construction efficiency and cost;
higher library yields will be achieved if a higher adapter concentration is used.

For Research Use Only. Not for use in diagnostic procedures. 5

KAPA HyperPlus Kit Technical Data Sheet
freezing and thawing of adapter stock solutions. Reaction Cleanups
• To accommodate different adapter concentrations • This protocol has been validated for use with either
within a batch of samples processed together, it is best KAPA Pure Beads or Agencourt AMPure XP (Beckman
to vary the concentrations of adapter stock solutions, Coulter). Solutions and conditions for DNA binding and
and dispense a fixed volume (5 µL) of each adapter. size selection may differ if other beads are used.
The alternative (using a single stock solution, and • Observe all the storage and handling recommendations
dispensing variable volumes of adapter into ligation for KAPA Pure Beads or AMPure XP. Equilibration to
reactions) is not recommended. room temperature is essential to achieve specified size
Post-ligation Processing distribution and yield of libraries.
• It is important to remove unligated adapter and/or • Beads will settle gradually; always ensure that they are
adapter-dimer molecules from the library prior to library fully resuspended before use.
amplification or cluster generation.
• To ensure optimal DNA recovery, it is critical that the
• The KAPA HyperPlus chemistry reduces adapter-dimer DNA and the KAPA Pure Beads are thoroughly mixed
formation, and enables efficient elimination of unused (by vortexing or extensive up-and-down pipetting)
adapter and adapter-dimer with a single post-ligation before the DNA binding incubation.
cleanup. The optimal bead to DNA ratio for libraries
• Bead incubation times are guidelines only, and may
prepared from fragmented dsDNA with a mode
be modified/optimized according to current protocols,
fragment length in the range of 150 – 350 bp is 0.8X.
previous experience, specific equipment and samples
This ratio may be modified to accommodate libraries
in order to maximize library construction efficiency and
prepared from longer DNA fragments or custom
throughput.
adapter designs, or to shift the mode fragment length
of adapter-ligated molecules. • The time required for complete capture of beads varies
according to the reaction vessel and magnet used. It
• The volume in which washed beads are resuspended
is important not to discard or transfer any beads with
after the post-ligation cleanup should be adjusted to
the removal or transfer of supernatant. Capture times
suit your chosen workflow:
should be optimized accordingly.
-- If proceeding directly to library amplification,
• The volumes of 80% ethanol used for bead washes may
determine the appropriate final volume in which
be adjusted to accommodate smaller reaction vessels
to elute the library DNA, keeping in mind that you
and/or limited pipetting capacity, but it is important
may wish to divert and/or reserve some of this
that the beads are entirely submerged during the wash
library material for archiving and/or QC purposes.
steps. Always use freshly prepared 80% ethanol.
Since a 50 µL library amplification reaction can
accommodate 20 – 24 µL template DNA, an elution • It is important to remove all ethanol before proceeding
volume of ~25 µL is recommended. with subsequent reactions. However, over-drying of
beads may make them difficult to resuspend, resulting
-- If proceeding with size selection, elute the library
in a dramatic loss of DNA. With optimized aspiration
DNA in a volume appropriate for the size selection
of ethanol, drying of beads for 3 – 5 min at room
method of choice. For the double-sided size
temperature should be sufficient. Drying of beads at
selection protocol described in Appendix 1, beads
37°C is not recommended.
must be resuspended in 55 µL of elution buffer.
• Where appropriate, DNA should be eluted from beads
• A second post-ligation cleanup (using a 1X or different
in elution buffer (10 mM Tris-HCl, pH 8.0 – 8.5). Elution
bead-to-DNA ratio) may be performed if post-ligation
of DNA in PCR-grade water is not recommended, as
or post-amplification analysis reveals unacceptable
DNA is unstable in unbuffered solutions. However,
levels of adapter and/or adapter-dimer carry-over after
libraries constructed for target capture must be eluted
the first cleanup. A second cleanup may be particularly
and stored in PCR-grade water to facilitate drying of
beneficial when libraries are prepared in PCR-free
DNA prior to probe hybridization. Purified DNA in elution
workflows for direct sequencing on Illumina instruments
buffer should be stable at 2°C to 8°C for 1 – 2 weeks,
that employ patterned flow cells. The sample volume
or at -15°C to -25°C for long-term storage. The long-
should be adjusted (with elution buffer) to at least
term stability of library DNA at -15°C to -25°C depends
50 µL for a second post-ligation cleanup. The adapter
on a number of factors, including library concentration.
concentration may also be optimized to eliminate carry-
Always use low DNA-binding tubes for long-term
over of adapter and/or adapter-dimer (and eliminate
storage, and avoid excessive freezing and thawing.
the need for a second post-ligation cleanup). However,
keep in mind that library construction is most efficient
when high adapter:insert molar ratios are used.

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

KAPA HyperPlus Kit Technical Data Sheet
Size Selection • The Library Amplification Primer Mix (10X) is designed
• Size selection requirements vary widely for different to eliminate or delay primer depletion during library
sequencing applications. If required, any commonly amplification reactions performed with KAPA HiFi
used bead- or gel-based size selection technique may HotStart ReadyMix. The primer mix is suitable for the
be integrated in the KAPA HyperPlus workflow. amplification of all Illumina libraries flanked by the P5
and P7 flow cell sequences. Primers are supplied at
• Size selection may be carried out at different points in a 10X concentration of 20 µM each, and have been
the overall workflow, for example after the post-ligation formulated as described below. User-supplied primer
cleanup, or after library amplification. mixes may be used in combination with incomplete or
• The standard protocol (pp. 12 – 14) does not include custom adapters. Please contact Technical Support at
size selection. Please refer to Appendix 1 (p. 15) for a [Link]/support for guidelines on the
detailed double-sided size selection protocol. formulation of user-supplied library amplification primers.
• Size selection inevitably leads to a loss of sample • To achieve the highest amplification efficiency and
material. These losses can be dramatic (60 – 95%), and avoid primer depletion, it is critical to use an optimal
may significantly increase the number of amplification concentration of high quality primers. Primers should
cycles required to generate sufficient material for the be used at a final concentration of 0.5 – 4 μM each.
next step in the process (capture or sequencing). The For libraries constructed from ≥100 ng input DNA, a
potential advantages of one or more size selection final concentration of at least 2 μM of each primer is
steps in a library construction workflow should be recommended.
weighed against the potential loss of library complexity, • Library amplification primers should be HPLC-purified
especially when input DNA is limited. A well-optimized and modified to include a phosphorothioate bond at
fragmentation protocol, especially for shorter insert the 3'-terminal of each primer (to prevent degradation
libraries and/or read lengths, may eliminate the need by the strong proofreading activity of KAPA HiFi
for size selection, thereby simplifying the library HotStart). Always store and dilute primers in a buffered
construction process and limiting sample losses. solution (e.g., 10 mM Tris-HCl, pH 8.0 – 8.5), and limit
• The Ligation Buffer contains high concentrations the number of freeze-thaw cycles. To achieve the latter,
of PEG 6000, which will interfere with efficient size store primers at 2°C to 8°C for short-term use, or as
selection and can affect the efficiency of other single-use aliquots at -15°C to -25°C.
size selection techniques if not removed. If size • In library amplification reactions (set up according to the
selection is performed after ligation, it is important recommended protocol), primers are typically depleted
to perform at least one bead-based cleanup prior before dNTPs. When DNA synthesis can no longer
to performing bead- or electrophoresis-based take place due to substrate depletion, subsequent
size selection. rounds of DNA denaturation and annealing result in the
• Over-amplification typically results in the observation separation of complementary DNA strands, followed by
of secondary, higher molecular weight peaks in imperfect annealing to non-complementary partners.
electrophoretic profiles of amplified libraries. These This presumably results in the formation of so-called
higher molecular weight peaks are artifacts of the “daisy chains” or “tangled knots”, comprising large
analysis, and typically contain authentic library assemblies of improperly annealed, partially double-
molecules of the appropriate length. To eliminate these stranded, heteroduplex DNA. These species migrate
artifacts, optimization of library amplification reaction slower and are observed as secondary, higher
parameters (cycle number and primer concentration), molecular weight peaks during the electrophoretic
rather than post-amplification size selection, is analysis of amplified libraries. However, they typically
recommended. Please refer to the next subsection for comprise library molecules of the desired length,
more information. which are individualized during denaturation prior
to cluster amplification or probe hybridization. Since
Library Amplification these heteroduplexes contain significant portions of
• KAPA HiFi HotStart, the enzyme provided in the KAPA single-stranded DNA, over-amplification leads to the
HiFi HotStart ReadyMix, is an antibody-based hot start under-quantification of library molecules with assays
formulation of KAPA HiFi DNA Polymerase—a novel employing dsDNA-binding dyes. qPCR-based library
B-family DNA polymerase engineered for increased quantifications methods, such as the KAPA Library
processivity and high fidelity. KAPA HiFi HotStart has Quantification assay, quantify DNA by denaturation
5'g3' polymerase and 3'g5' exonuclease (proofreading) and amplification, thereby providing an accurate
activity, but no 5'g3' exonuclease activity. The strong measure of the amount of adapter-ligated molecules in
3'g5' exonuclease activity results in superior accuracy a library—even if the library was over-amplified.
during DNA amplification. The error rate of KAPA HiFi
HotStart is 2.8 x 10-7 errors/base, equivalent to 1 error
in 3.5 x 106 nucleotides incorporated.

For Research Use Only. Not for use in diagnostic procedures. 7

KAPA HyperPlus Kit Technical Data Sheet
• Please refer to the KAPA NGS Library Preparation Please refer to Table 6 for the number of cycles
Technical Guide for a more detailed discussion recommended to obtain approximately 1 µg of DNA
of factors that can affect the efficiency of library from 500 pg – 500 ng of adapter-ligated DNA, or
amplification, and the impact of over-amplification on contact Technical Support at [Link]/
library quantification. support regarding a calculator designed to assist with
these calculations. Please note that the actual optimal
• Excessive library amplification can result in other
number of amplification cycles may be 1 – 3 cycles
unwanted artifacts such as amplification bias, PCR
higher or lower, depending on the sample type and size
duplicates, chimeric library inserts and nucleotide
distribution of the input DNA.
substitutions. The extent of library amplification
should therefore be limited as much as possible, • Depending on the amount of library material required
while ensuring that sufficient material is generated for for your application, it may be possible to omit library
QC and downstream processing (e.g., target capture amplification. In such cases, it is important to ensure
or sequencing). that your adapters are designed to support sample
indexing (where required), cluster amplification and
• If cycled to completion (not recommended), one 50 µL
sequencing. Omitting library amplification further
library amplification PCR—performed as described
streamlines the workflow and reduces overall library
in the Library Construction Protocol (step 5)—can
preparation time to ≤1.5 hrs. The high conversion
produce 8 – 10 µg of amplified library. To minimize
efficiency achievable with the KAPA HyperPlus Kit
over-amplification and its associated, undesired
enables PCR-free workflows from as little as 50 ng of
artifacts, the number of amplification cycles should be
input DNA. KAPA HyperPlus Kits without amplification
tailored to produce the optimal amount of final library
reagents (KK8511, KK8513, KK8515) are available for
required for downstream processes. This is typically
PCR-free workflows.
in the range of 250 ng – 1.5 µg. Table 5 provides
recommended cycle numbers for libraries prepared Table 6. Theoretical number of cycles required to
from high-quality input DNA, to obtain approximately obtain approximately 1 µg of amplified library DNA from
100 ng or 1 µg of amplified library. 500 pg – 500 ng of adapter-ligated library DNA*

Table 5. Recommended cycle numbers to generate 100 ng or Amount of adapter-ligated Number of cycles required to
1 µg of amplified DNA from 1 ng – 1 µg of input DNA DNA in amplification rxn generate 1 µg of library DNA
500 ng 1–2
Input into library Number of cycles required to generate
100 ng 3–4
construction 100 ng library 1 µg library
50 ng 5–6
1 µg 0* 0 – 1*
10 ng 7–8
500 ng 0* 2–3
5 ng 8–9
250 ng 0 – 1* 3–5
1 ng 11 – 12
100 ng 0 – 2* 5–6
500 pg 12 – 13
50 ng 3–5 7–8
*Guidelines are based on amplification with KAPA HiFi HotStart ReadyMix and the
25 ng 5–6 8 – 10 Library Amplification Primer Mix, and library quantification with the qPCR-based
KAPA Library Quantification Kit.
10 ng 7–9 11 – 13
5 ng 9 – 11 13 – 14 Evaluating the Success of Library Construction
2.5 ng 11 – 13 14 – 16 • Your specific library construction workflow should be
1 ng 13 – 15 17 – 19 tailored and optimized to yield a sufficient amount
*When using incomplete adapters, a minimum number of amplification cycles of adapter-ligated molecules of the desired size
(1 – 3) may be required to complete adapter sequences for the next step in the distribution for the next step in the process (e.g., target
process (target capture or sequencing), irrespective of whether a sufficient amount
of library is available after ligation. The number of cycles needed depends on the capture or sequencing), as well as for library QC and
specific adapter and amplification primer design. archiving purposes.

• The quantification of adapter-ligated libraries (prior • While it is possible to remove aliquots of the
to library amplification) can greatly facilitate the fragmentation reaction product for analysis in
optimization of library amplification parameters, the integrated fragmentation/library construction
particularly when a library construction workflow is workflow, it is most productive to assess the outcome
first established. With the KAPA Library Quantification of fragmentation once the entire KAPA HyperPlus
Kit, the amount of template DNA (adapter-ligated workflow has been completed—for the following
molecules) available for library amplification can be reasons:
determined accurately. From there, the number of -- It is difficult and disruptive to process low-volume
amplification cycles needed to achieve a specific yield aliquots in a way that is fully representative of the
of amplified library can be predicted theoretically. final library.

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

KAPA HyperPlus Kit Technical Data Sheet
-- Fragmentation profiles for low-input samples The KAPA Library Quantification Kit provides the only
(1 – 10 ng into fragmentation) may not be informative, reliable means for quantifying libraries at different
even when high-sensitivity assays are used. stages of the workflow, and libraries produced in PCR-
free workflows, as:
-- The final size distribution of libraries prepared from
FFPE samples is typically smaller than expected -- it only quantifies those molecules with two adapters
based on the size distribution after fragmentation in the correct configuation for cluster amplification
and adapter length. This is a common phenomenon, and sequencing, and
attributable to the inability of high-fidelity DNA
-- measurements are not affected by library over-
polymerases used in library amplification to efficiently
amplification (see Important Parameters: Library
amplify damaged DNA, particularly templates that
Amplification, p. 7).
contain deaminated or oxidized bases.
• Once a library construction workflow has been
• Please refer to Appendix 2 (p. 16) for guidelines on how optimized, and consistently yields the desired amount
to systematically optimize fragmentation parameters of amplified library of the requisite size distribution,
for your specific samples. it is typically not necessary to perform in-process
quality control. However, qPCR-based quantification
• The size distribution of pre-capture or final libraries
of libraries after the Post-ligation Cleanup (prior to
should be confirmed with an electrophoretic method.
Library Amplification) can provide useful data for
A LabChip GX, GXII, or GX Touch (PerkinElmer),
optimization or troubleshooting. Quantification at this
Bioanalyzer or TapeStation (Agilent Technologies),
stage allows you to assess the efficiency of:
Fragment Analyzer (Advanced Analytical) or similar
instrument is recommended over conventional gels. -- the core library construction process (fragmentation
Typical electrophoretic profiles for libraries prepared to ligation), by determining the percentage of input
with the KAPA HyperPlus Kit are given in Figure 1 on DNA converted to adapter-ligated molecules, and
the next page.
-- library amplification with the selected number of
• Please note that libraries prepared with “forked” cycles, based on the actual amount of template
adapters in PCR-free workflows will appear to have DNA used in the PCR.
a longer than expected mode fragment length, and/
The availability of quantification data before and after
or may display a broad or bimodal size distribution
library amplification allows the two major phases of
when analyzed electrophoretically (see Figure 1). The
the library construction process to be evaluated and
difference in overall appearance and fragment size
optimized independently to achieve the desired yield
distribution of an unamplified vs. the corresponding
of amplified library.
amplified library varies, and depends on the adapter
design and electrophoretic system used. To accurately • If size selection is performed at any stage, qPCR
determine the size distribution of an unamplified library, quantification before and after size selection may also
an aliquot of the library may be subjected to a few be helpful to define the relative benefit of size selection,
cycles of amplification prior to electrophoretic analysis, and to determine the loss of material associated with
to ensure that all adapter-ligated molecules are fully the process.
double-stranded. Alternatively, size information may
• Electrophoretic evaluation of libraries after the post-
be obtained by electrophoretic analysis of library
ligation cleanup/before library amplification may be
quantification products generated with the KAPA
informative, but remember that the apparent mode
Library Quantification Kit (see below).
fragment length and size distribution will be inaccurate
• KAPA Library Quantification Kits for Illumina platforms due to the retardation of non-complementary adapter
are recommended for qPCR-based quantification of regions, as outlined above and illustrated in Figure 1.
libraries generated using the KAPA HyperPlus workflow.
These kits employ primers based on the Illumina flow
cell oligos, and can be used to quantify libraries that:
-- are ready for flow-cell amplification, and/or
-- were constructed with full-length adapters, once
ligation has been completed, i.e., after the post-
ligation cleanup, after the (pre-capture) amplification
cleanup, or before/after post-ligation or post-
amplification size selection.

For Research Use Only. Not for use in diagnostic procedures. 9

KAPA HyperPlus Kit Technical Data Sheet

[FU] [FU]

A Final, amplified library

200 B Final, amplified library

Final, amplified library

150
Fragmented (size selected)
DNA 150
Fragmented
DNA
100
Post-ligation 100
Post-ligation
library library

50
50

0 0

35 100 150 200 300 400 500 600 1000 2000 10380 [bp] 35 100 150 200 300 400 500 600 1000 2000 10380 [bp]

Figure 1. Examples of libraries prepared with the KAPA HyperPlus Kit

Input DNA (100 ng high-quality human genomic DNA) was fragmented at 37°C for 30 min (A) or 10 min (B), to achieve a mode fragment length of ~150 bp or ~350 bp,
respectively. Libraries were prepared as described in the Library Construction Protocol (pp. 12 – 14), using the recommended adapter:insert molar ratio. Larger-insert libraries
(B) were prepared in duplicate. One library was subjected to double-sided size selection after the Post-ligation Cleanup, as described in Appendix 1: Size Selection (p. 15),
whereas the other was not. Electropherograms were generated with a Bioanalyzer 2100 High Sensitivity DNA Kit. DNA concentrations were normalized prior to analysis and
are not reflective of the actual DNA concentrations at different stages of the process. Electrophoretic profiles for fragmented DNA were generated in a “standalone” workflow,
whereby the protocol was terminated after fragmentation, and reaction products purified using a 2X bead-based cleanup.
After ligation, the non-complementary ends of full-length, “forked” adapters retard the migration of library fragments in gel matrices, leading to a larger than expected size
distribution. The difference between the actual and apparent mode fragment length of unamplified, adapter-ligated libraries depends on the adapter design and electrophoretic
system used, and can be much more pronounced than observed here. Size selection results in a much narrower final library size distribution, but at the cost of a significant
amount of library material.

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

KAPA HyperPlus Kit Technical Data Sheet
Process Workflow

Recommended QC Metrics
See Table 1 (p. 4) for recommended
inputs of different types of DNA for
different sequencing applications. Concentration and Q-ratio of input DNA
For samples containing EDTA: perform a dsDNA sample (35 µL) (KAPA hgDNA Quantification and QC Kit)
bead-based cleanup with KAPA Pure Beads for FFPE DNA only
or include the appropriate concentration of
Conditioning Solution (Table 2, p. 4).

Enzymatic Fragmentation (50 µL)

5 – 40 min at 37°C
Transfer to 4°C
(step 1)

End Repair and A-tailing (60 µL)

30 min at 65°C
(step 2)

See Table 3 (p. 5) for recommended

adapter stock concentrations.

Adapter Ligation (110 µL)

15 min at 20°C
(step 3)

Post-ligation Cleanup (198 µL)

0.8X Bead-based Cleanup
Size selection—employing KAPA Pure Beads (step 4) Concentration of adapter-ligated libraries
or an electrophoretic method—may be (KAPA Library Quantification Kit)
incorporated at this point, if appropriate.
Adapter-ligated libraries diluted in the range
PCR-free workflows end here. of 1/500 to 1/10,000 usually fall within the
Library Amplification (50 µL) dynamic range of the assay

(step 5)

Post-amplification
Cleanup (50 µL)
1X Bead-based Cleanup
(step 6) Electrophoretic profile of amplified libraries
Size selection—employing KAPA Pure Beads Concentration of amplified libraries
or an electrophoretic method—may be (KAPA Library Quantification Kit)
incorporated at this point, if appropriate.
Amplified libraries diluted in the range of
1/10,000 to 1/200,000 usually fall within the
Target Capture or Sequencing dynamic range of the assay

For Research Use Only. Not for use in diagnostic procedures. 11

KAPA HyperPlus Kit Technical Data Sheet
Library Construction Protocol 1.3 A
 ssemble each fragmentation reaction on ice by
adding the components in this order:
Notes:
• First-time users should refer to Appendix 2: Component Volume
Optimization of Fragmentation Parameters (p. 16) Double-stranded DNA (with
35 µL
before trying this kit, as standard fragmentation Conditioning Solution, if needed)
parameters may not result in the optimal size distribution KAPA Frag Buffer (10X)* 5 µL
for libraries prepared from your specific DNA samples. KAPA Frag Enzyme* 10 µL
Precious samples should not be used when evaluating Total volume: 50 µL
this kit. Instead, parameters should be optimized with
*The KAPA Frag Buffer and Enzyme may be pre-mixed and kept on ice
a non-precious, bulk DNA sample that is representative prior to reaction setup, and dispensed as a single solution. Please note
of the actual samples to be processed. the volume of buffer is less than the volume of enzyme in this reaction.

• If your DNA samples contain EDTA, please consult the 1.4 V

 ortex gently and spin down briefly. Return the
Appendix 2: Handling of DNA Samples Containing plate/tube(s) to ice. Proceed immediately to the
EDTA (p. 16), as well as Important Parameters: Input next step.
DNA (p. 4) before starting this protocol. 1.5 Incubate in a thermocycler, pre-cooled to 4°C and
• This protocol does not include size selection. Please programmed as outlined below. A heated lid is not
refer to Appendix 1 (p. 15) for a detailed double-sided required for this step. If used, set the temperature
size selection protocol that may be included after of the heated lid to ≤50°C.
ligation or after amplification.
Step Temp Time
• Always ensure that KAPA Pure Beads are fully equilibrated
to room temperature and fully resuspended before use. Pre-cool block 4°C N/A
Fragmentation 37°C See table below
1. Enzymatic Fragmentation HOLD 4°C ∞
If the DNA samples contain EDTA, perform a

3X bead-based cleanup with KAPA Pure Beads Mode fragment Incubation time Optimization
length at 37°C* range
to remove EDTA prior to fragmentation. Please
refer to the KAPA Pure Beads Technical Data 600 bp 5 min 3 – 10 min
Sheet for a detailed DNA cleanup protocol. 350 bp 10 min 5 – 20 min
Alternatively, prepare a sufficient volume of 200 bp 20 min 10 – 25 min
appropriately diluted Conditioning Solution 150 bp 30 min 20 – 40 min
(5 μL per DNA sample, plus excess). Refer to *These parameters are a good starting point for high-quality genomic
Table 2 (p. 4) for guidelines on the dilution of the DNA. Please refer to Appendix 2: Optimization of Fragmentation
Parameters (p. 16) for guidelines on how to optimize fragmentation
Conditioning Solution. time and temperature. If incubation times longer than the recommended
range are needed, samples likely contain inhibitors which impact
1.1 D
 ilute the amount of dsDNA to be used for library the fragmentation efficiency. Bead-based DNA cleanup, prior to
fragmentation, is recommended over longer fragmentation times.
construction as follows:
• If the DNA preparation does not contain EDTA, 1.6 T
 ransfer reactions to ice, and proceed immediately
dilute in 10 mM Tris-HCl (pH 8.0 – 8.5) in a to End Repair and A-tailing (step 2).
total of 35 µL
2. End Repair and A-tailing
• If the DNA preparation does contain EDTA,
dilute in the EDTA-containing buffer in which 2.1 In the same plate/tube(s) in which enzymatic
samples are currently suspended, in a total fragmentation was performed, assemble each end
of 30 μL. To each reaction with 30 μL of repair and A-tailing reaction as follows:
EDTA-containing DNA, add 5 μL of diluted
Component Volume
Conditioning Solution.
Fragmented, double-stranded DNA 50 µL
1.2 Mix by gentle vortexing or pipetting up and down.
End Repair & A-Tailing Buffer* 7 µL
End Repair & A-Tailing Enzyme Mix* 3 µL
Total volume: 60 µL
*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.

2.2 V
 ortex gently and spin down briefly. Return the
reaction plate/tube(s) to ice. Proceed immediately
to the next step.

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

KAPA HyperPlus Kit Technical Data Sheet
2.3 Incubate in a thermocycler programmed as 4. Post-ligation Cleanup
outlined below. A heated lid is required for this
4.1 In the same plate/tube(s), perform a 0.8X bead-
step. If possible, set the temperature of the heated
based cleanup by combining the following:
lid to ~85°C (instead of the usual 105°C).
Component Volume
Step Temp Time
Adapter ligation reaction product 110 µL
End repair and A-tailing 65°C* 30 min
KAPA Pure Beads 88 µL
HOLD 4°C** ∞
Total volume: 198 µL
*Both the fragmentation and end repair enzymes are inactivated at 65°C.
When reactions are set up according to recommendations, additional
fragmentation should be negligible. The brief period of end repair is
sufficient for enzymatically fragmented DNA. 4.2 M
 ix thoroughly by vortexing and/or pipetting up
**If proceeding to the adapter ligation reaction setup without any delay, and down multiple times.
the reaction may be cooled to 20°C instead of 4°C.
4.3 Incubate the plate/tube(s) at room temperature for
2.4 Proceed immediately to Adapter Ligation (step 3). 5 – 15 min to bind DNA to the beads.
3. Adapter Ligation 4.4 P
 lace the plate/tube(s) on a magnet to capture the
beads. Incubate until the liquid is clear.
3.1 D
ilute adapter stocks to the appropriate
concentration, as outlined in Table 4 (p. 5). 4.5 Carefully remove and discard the supernatant.
3.2 In the same plate/tube(s) in which end repair and 4.6 K
 eeping the plate/tube(s) on the magnet, add
A-tailing was performed, assemble each adapter 200 µL of 80% ethanol.
ligation reaction as follows: 4.7 Incubate the plate/tube(s) on the magnet at room
Component Volume temperature for ≥30 sec.
End repair and A-tailing reaction 4.8 Carefully remove and discard the ethanol.
60 µL
product
4.9 K
 eeping the plate/tube(s) on the magnet, add
Adapter stock
5 µL 200 µL of 80% ethanol.
(diluted as per Table 4 on p. 5)
PCR-grade water* 5 µL 4.10 Incubate the plate/tube(s) on the magnet at room
Ligation Buffer* 30 µL temperature for ≥30 sec.
DNA Ligase* 10 µL 4.11 C
 arefully remove and discard the ethanol. Try to
Total volume: 110 µL remove all residual ethanol without disturbing
the beads.
*The water, buffer and ligase enzyme should preferably be premixed
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
4.12 D
 ry the beads at room temperature for 3 – 5 min,
-15°C to -25°C. or until all of the ethanol has evaporated. Caution:
over-drying the beads may result in reduced yield.
3.3 Mix thoroughly and centrifuge briefly.
4.13 Remove the plate/tube(s) from the magnet.
3.4 Incubate at 20°C for 15 min.
4.14 Resuspend the beads:
 ote: to achieve higher conversion rates and library
N
• in 25 µL of elution buffer (10 mM Tris-HCl,
yields, particularly for low-input samples, consider
pH 8.0 – 8.5) to proceed with Library
increasing the ligation time to a maximum of 4 hrs
Amplification (step 5), or
at 20°C or overnight at 2°C to 8°C. Please note
that longer ligation times may lead to increased • in 55 µL of elution buffer (10 mM Tris-HCl,
levels of adapter-dimer. Adapter concentrations pH 8.0 – 8.5) to proceed with double-sided
may have to be optimized if ligation times are size selection (Appendix 1).
extended significantly.
4.15 Incubate the plate/tube(s) at room temperature for
3.5 P
roceed immediately to Post-ligation Cleanup 2 min to elute DNA off the beads.
(step 4).
4.16 P
 lace the plate/tube(s) on a magnet to capture the
beads. Incubate until the liquid is clear.
4.17 Transfer the clear supernatant to a new plate/tube(s):
• t o proceed with Library Amplification (step 5),
transfer 20 µL of supernatant, or
• to proceed with double-sided size selection
(Appendix 1), transfer 50 µL of supernatant.

For Research Use Only. Not for use in diagnostic procedures. 13

KAPA HyperPlus Kit Technical Data Sheet
5. Library Amplification 6. Post-amplification Cleanup
 ote: Please refer to Important Parameters:
N 6.1 In the library amplification plate/tube(s), perform a
Library Amplification (p. 7) and the KAPA NGS 1X bead-based cleanup by combining the following:
Library Preparation Technical Guide for more
information on optimizing library amplification. Component Volume
Library amplification reaction product 50 µL
5.1 A
 ssemble each library amplification reaction
as follows: KAPA Pure Beads 50 µL
Total volume: 100 µL
Component Volume
KAPA HiFi HotStart ReadyMix (2X) 25 µL 6.2 M
 ix thoroughly by vortexing and/or pipetting up
Library Amplification Primer Mix (10X)* 5 µL and down multiple times.
Adapter-ligated library 20 µL 6.3 Incubate the plate/tube(s) at room temperature for
Total volume: 50 µL 5 – 15 min to bind DNA to the beads.
*Or other, suitable 10X library amplification primer [Link] recommended 6.4 P
 lace the plate/tube(s) on a magnet to capture the
final concentration of each primer in the library amplification reaction is
0.5 – 4 µM. Also refer to Important Parameters: Library Amplification beads. Incubate until the liquid is clear.
(p. 7).
6.5 Carefully remove and discard the supernatant.
5.2 Mix thoroughly and centrifuge briefly. 6.6 K
 eeping the plate/tube(s) on the magnet, add
5.3 Amplify using the following cycling protocol: 200 µL of 80% ethanol.
6.7 Incubate the plate/tube(s) on the magnet at room
Step Temp Duration Cycles
temperature for ≥30 sec.
Initial
98°C 45 sec 1 6.8 Carefully remove and discard the ethanol.
denaturation

Denaturation 98°C 15 sec Minimum 6.9 K

 eeping the plate/tube(s) on the magnet, add
number 200 µL of 80% ethanol.
required
Annealing* 60°C 30 sec 6.10 Incubate the plate/tube(s) on the magnet at room
for optimal
amplification temperature for ≥30 sec.
Extension 72°C 30 sec (Table 5 or 6)
6.11 C
 arefully remove and discard the ethanol. Try to
Final remove all residual ethanol without disturbing
72°C 1 min 1
extension the beads.
HOLD 4°C ∞ 1
6.12 D
 ry the beads at room temperature for 3 – 5 min,
*Optimization of the annealing temperature may be required for non-
standard (i.e., other than Illumina TruSeq) adapter/primer combinations. or until all of the ethanol has evaporated. Caution:
over-drying the beads may result in reduced yield.
5.4 P
 roceed directly to Post-amplification Cleanup 6.13 Remove the plate/tube(s) from the magnet.
(step 6).
6.14 T
 horoughly 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.
6.15 Incubate the plate/tube(s) at room temperature for
2 min to elute DNA off the beads.
6.16 P
 lace the plate/tube(s) on a magnet to capture the
beads. Incubate until the liquid is clear.
6.17 T
ransfer 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.

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

KAPA HyperPlus Kit Technical Data Sheet
Appendix 1: Size Selection
Any commonly used size selection technique (e.g., the double-sided size selection described here, or an electrophoretic
method) may be integrated into the KAPA HyperPlus library construction workflow. Size selection should preferably be
carried out after the post-ligation cleanup, or after library amplification. Whether or not size selection is performed, which
technique is used, and at what stage of the library construction process it is performed, depends on the nature of the
sample, input into library construction, and the sequencing application and read length. For more information on size
selection, refer to Important Parameters: Size Selection (p. 7).
The double-sided size selection protocol outlined in this appendix is designed for the selection of library molecules
(inclusive of adapter) in the range of 250 – 450 bp. To obtain a population of shorter or longer molecules, the protocol may
be modified as follows:

Upper size limit Modification Lower size limit Modification

Increase Decrease the ratio of the first cut Increase Decrease the ratio of the second cut*
Decrease Increase the ratio of the first cut Decrease Increase the ratio of the second cut*
*The second size cut should be performed with at least 0.2 volumes of KAPA Pure Beads reagent. Please note that the volume of KAPA Pure Beads needed for the second cut
is calculated relative to the volume of the DNA at the start of the size selection procedure, not the volume of the DNA-containing supernatant transferred after the first cut. DNA
recovery is dramatically reduced if the difference between first and second cuts is less than ~0.2 volumes. To increase the amount of DNA recovered, >0.2 volumes of KAPA
Pure Beads may be used for the second cut, but note that this may result in the recovery of smaller library fragments and/or a broader size distribution. For more information
on double-sided size selection, please refer to the KAPA NGS Library Preparation Technical Guide, or visit [Link]/support for technical support.

A1.1 P
 erform the first (0.7X) size cut (to exclude library A1.9 P
 lace the plate/tube(s) on a magnet to capture the
molecules larger than ~450 bp) by combining the beads. Incubate until the liquid is clear.
following:
A1.10 C
 arefully remove and discard the supernatant,
Component Volume which contains library molecules smaller than
~250 bp.
DNA to be size selected 50 µL
KAPA Pure Beads 35 µL A1.11 K
 eeping the plate/tube(s) on the magnet, add
Total volume per well/tube: 85 µL 200 µL of 80% ethanol.
A1.12 Incubate the plate/tube(s) on the magnet at room
A1.2 M
 ix thoroughly by vortexing and/or pipetting up temperature for ≥30 sec.
and down multiple times.
A1.13 Carefully remove and discard the ethanol.
A1.3 Incubate the plate/tube(s) at room temperature for
5 – 15 min to bind library molecules larger than A1.14 K
 eeping the plate/tube(s) on the magnet, add
~450 bp to the beads. 200 µL of 80% ethanol.

A1.4 P
 lace the plate/tube(s) on a magnet to capture the A1.15 Incubate the plate/tube(s) on the magnet at room
beads. Incubate until the liquid is clear. temperature for ≥30 sec.

A1.5 C
 arefully transfer ~80 µL of supernatant containing A1.16 C
 arefully remove and discard the ethanol. Try to
library molecules smaller than ~450 bp to a new remove all residual ethanol without disturbing
plate/tube. It is critical that no beads are transferred the beads.
with the supernatant. Discard the plate/tube(s) A1.17 D
 ry the beads for 3 – 5 min at room temperature,
with the beads to which library molecules larger or until all of the ethanol has evaporated. Caution:
than ~450 bp were bound. over-drying the beads may result in reduced yield.
A1.6 P
 erform the second size cut (0.9X), to retain library A1.18 Remove the plate/tube(s) from the magnet.
molecules >250 bp) by combining the following:
A1.19 T
 horoughly resuspend the beads in the required
Component Volume volume of elution buffer (10 mM Tris-HCl,
Supernatant from first size cut 80 µL pH 8.0 – 8.5).
KAPA Pure Beads 10 µL A1.20 Incubate the plate/tube(s) at room temperature for
Total volume per well/tube: 90 µL 2 min to elute DNA off the beads.
A1.21 P
 lace the plate/tube(s) on a magnet to capture the
A1.7 M
 ix thoroughly by vortexing and/or pipetting up beads. Incubate until the liquid is clear.
and down multiple times.
A1.22 T
 ransfer the clear supernatant with size-selected
A1.8 Incubate the plate/tube(s) at room temperature for DNA to a new plate/tube(s) and proceed with the
5 – 15 min to bind library molecules larger than next step in your workflow, or store DNA at 2°C to
~250 bp to the beads. 8°C for 1 – 2 weeks, or at -15°C to -25°C.

For Research Use Only. Not for use in diagnostic procedures. 15

KAPA HyperPlus Kit Technical Data Sheet
Appendix 2: Optimization of Fragmentation • All samples will now contain the same final EDTA
Parameters concentration once diluted to 50 µL for fragmentation.
This concentration is:
Fragmentation guidelines provided in the Library
Construction Protocol: Enzymatic Fragmentation (step 1) EDTA concentration in TE buffer x (30 µL/50 µL)
may not result in the optimal library size distribution for your = 1 mM x (30 µL/50 µL) = 0.6 mM
specific DNA samples. For this reason, precious samples
should not be used when evaluating the KAPA HyperPlus • Make a 2.2-fold dilution of the Conditioning Solution
Kit for the first time. Instead, fragmentation parameters (as per Table 3 on p. 4), and follow the Library
should be optimized in the context of the KAPA HyperPlus Construction Protocol (second half of step 1.1).
workflow, using a non-precious, bulk DNA sample that is If you are unsure about the presence or concentration
representative of the actual samples to be processed. of EDTA in your DNA samples, remove the EDTA by
The information in this Appendix should be considered performing a column- or bead-based purification or buffer
during the experimental design for your evaluation of the exchange prior to enzymatic fragmentation, or follow the
KAPA HyperPlus Kit. procedure outlined below:

Quantification of Input DNA • Set up a series of test reactions with the appropriate
amount of input DNA, and different final concentrations
Although the KAPA Frag enzymatic fragmentation system
of Conditioning Solution.
is less sensitive to DNA input than tagmentation-based
library construction methods, it is recommended that input • Include at least one reaction with the same input of
DNA be quantified. PicoGreen/Qubit is recommended control DNA known to be EDTA-free. The control DNA
for the quantification of high-quality DNA, whereas the should preferably be of the same type and quality as
KAPA hgDNA Quantification and QC Kit provides both the test samples.
concentration and quality information for FFPE DNA. • Fragment the DNA using the appropriate parameters,
Handling of DNA Samples Containing EDTA as outlined in the Library Construction Protocol:
If the DNA samples contain EDTA, perform a 3X bead- Enzymatic Fragmentation (step 1). Complete the
based cleanup with KAPA Pure Beads to remove EDTA prior library construction process, and compare library size
to fragmentation. Please refer to the KAPA Pure Beads distributions for the test and control samples using an
Technical Data Sheet for a detailed DNA cleanup protocol. electrophoretic system (see Important Parameters:
Evaluating the Success of Library Construction, p. 8).
Alternatively, the Conditioning Solution may be used to
neutralize EDTA prior to fragmentation. This strategy is • Titrate the final concentration of Conditioning Solution
recommended as a first approach for precious FFPE DNA in the reaction until the test samples yield similar
samples of variable quality. fragmentation profiles as the EDTA-free control sample,
or until the desired library size distribution has been
Since EDTA in DNA preparations is usually introduced achieved.
via elution buffers used in the final stages of the DNA
extraction or purification process, the concentration • A two-step strategy may be the best. Start with 3 – 4 test
of EDTA is typically known (e.g., 1 mM for standard TE samples covering a broad range of final Conditioning
buffer and 0.1 mM for “low-EDTA” TE buffer). If this is the Solution concentrations, then perform a finer titration
case, and your samples are of similar concentration (i.e., a over a narrower concentration range.
constant volume of DNA is used for library construction),
simply refer to Table 3 (p. 4) for the appropriate dilution of Optimization of Fragmentation Time
Conditioning Solution, and follow the Library Construction The fragmentation guidelines in the Library Construction
Protocol (step 1.1). Protocol: Enzymatic Fragmentation (step 1) are a good
starting point for high-quality genomic DNA. When
If you know the composition of the EDTA-containing buffer evaluating the KAPA HyperPlus Kit for the first time, it is
used for DNA purification, but your samples span a wide recommended that you proceed as follows:
concentration range (i.e., variable volumes will be used
to achieve the desired input into library construction), • Set up at least three replicate reactions with the
samples should be normalized in the same EDTA- desired input of a non-precious, bulk sample that is
containing buffer used for DNA purification. representative of the actual samples to be processed.
For example: • Select the most appropriate fragmentation time (for
the desired mode fragment length) from the third table
• If your DNA samples are in TE buffer, and your input in the Library Construction Protocol (step 1.5, p. 12).
into library construction is 100 ng, dilute 100 ng of each Perform one reaction with that time, and one reaction
sample into a final volume of 30 µL (i.e., to 3.33 ng/µL) each with either a slightly shorter or slightly longer
using TE buffer. fragmentation time within the optimization range.
Increments of 3 – 5 min are recommended.

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

KAPA HyperPlus Kit Technical Data Sheet
• Complete the library construction process, and Optimization of Fragmentation Temperature
evaluate the size distribution of the final libraries The standard fragmentation temperature is 37°C. If you
electrophoretically. are fragmenting high-quality genomic DNA, any other
-- If the mode fragment length is too long, increase high-complexity DNA sample, or FFPE DNA to a mode
the fragmentation time in increments of 2 – 5 min fragment length <500 bp, it is unlikely that you will have to
until the optimal final library distribution is achieved. change or optimize the fragmentation temperature.

-- If the mode fragment length is too short, reduce the Low-complexity samples (e.g., small viral genomes,
fragmentation time in increments of 2 – 5 min until plasmids, long amplicons and cDNA) may, however, be
the optimal final library distribution is achieved. over-fragmented at 37°C, even with short incubation
times. The likelihood of over-fragmentation depends on
• Further fine-tuning (plus or minus 1 – 2 min) may be the nature, molecular weight/length of the input DNA, the
necessary if the fragmentation time is relatively short desired size distribution after fragmentation and, to a lesser
(10 min or less). If this is the case, consider optimizing degree, the DNA input into fragmentation. For example,
the fragmentation temperature (see below). 100 ng of a 1.8 kb PCR product will yield a similar mode
A similar strategy may be employed to optimize the fragment length (~300 bp) as 100 ng E. coli or human
fragmentation time for FFPE samples, remembering that genomic DNA when fragmented at 37°C for 10 min,
lower quality samples may benefit from slightly longer whereas 1 ng of a 1 kb PCR product will be fragmented to
fragmentation times. For FFPE samples: a mode size <250 bp using the same parameters.
• Set up 4 – 5 replicate reactions with the desired input of To determine the optimal fragmentation parameters for
a non-precious, bulk sample that is representative of the low-complexity samples, or high-complexity samples
actual samples to be processed. This sample may have when the desired mode fragment length is >500 bp:
to be generated by pooling a few individual samples. • Set up four replicate reactions with a non-precious,
• Select the fragmentation time corresponding to the bulk sample that is representative of the actual samples
desired mode fragment length from the third table in to be processed.
the Library Construction Protocol (step 1.5, p. 12). • Fragment two of the samples at 37°C, for 5 min and
Use that as the minimum fragmentation time, and 10 min, respectively. Repeat these fragmentations with
increase the incubation time at 37°C by 5 min for each the other two samples, but at 30°C.
additional replicate.
• Complete the library construction process, and
• Complete the library construction process, evaluate evaluate the size distribution of the final libraries
the size distribution of the final libraries, and fine-tune electrophoretically.
fragmentation time if needed, as described above.
-- If the mode fragment length obtained with a 10 min
With respect to FFPE samples, please note the following: incubation at 37°C is too long, continue optimizing
• Electrophoretic profiles of FFPE samples, generated (increasing) the fragmentation time at 37°C.
during sample QC prior to fragmentation, are not -- If the mode fragment length obtained with a 10 min
always good predictors of library and sequence quality. incubation at 30°C is too long, but 5 min at 37°C
Samples that appear to consist of high-molecular resulted in over-fragmentation, continue optimizing
weight DNA may not yield libraries of significantly (increasing) the fragmentation time at 30°C.
better quality than samples that appear to be degraded.
The KAPA hgDNA Quantification and QC Kit provides -- If a 5 min incubation at 30°C resulted in over-
a qPCR-based assay for assessment of FFPE DNA fragmentation, perform a second set of reactions
quality. Quality scores (Q-ratios) determined with this (e.g., for 5 min, 10 min, 15 min, and 20 min) at 25°C,
assay have been shown to correlate with the success and fine-tune the fragmentation time if needed.
of library construction.
• The mode fragment length of an amplified FFPE library
is typically shorter than than expected based on the
size distribution after fragmentation and adapter
length. This is a common phenomenon, attributable
to the inability of high-fidelity DNA polymerases used
in library amplification to efficiently amplify damaged
DNA, particularly templates that contain deaminated or
oxidized bases. For this reason, it is not productive to try
and optimize fragmentation parameters independently
of the rest of the library construction process when
using the KAPA HyperPlus workflow.

For Research Use Only. Not for use in diagnostic procedures. 17

KAPA HyperPlus Kit Technical Data Sheet
Appendix 3: Library Construction Guidelines Post-ligation Processing
for the Roche SeqCap EZ Target Capture • Perform the 0.8X post-ligation cleanup as outlined
System in the Library Construction Protocol: Post-ligation
Cleanup (step 4). Resuspend the beads with adapter-
The KAPA HyperPlus Kit may be used (instead of a KAPA ligated DNA in 55 µL elution buffer, and recover 50 µL
HTP/LTP Library Preparation Kit) for the construction of of supernatant.
pre-capture libraries in the SeqCap EZ workflow. The
KAPA HyperPlus Kit offers a more streamlined protocol • Perform a 0.7X – 0.9X double-sided size selection with
that does not require mechanical fragmentation, shorter KAPA Pure Beads, as outlined in Appendix 1 (p. 15).
turnaround times and improved performance with low- • Resuspend beads with size-selected, adapter-ligated
input and challenging samples, such as FFPE and cell-free/ DNA in 22 – 25 µL elution buffer, and recover 20 µL of
circulating tumor DNA. This quick protocol guide provides supernatant.
specific guidelines for such an integrated workflow.
Library Construction Pre-capture Library Amplification (LM-PCR)
Detailed instructions for specific steps or processes are • Amplify 20 µL of each pre-capture library, using the
included below. reaction setup and cycling parameters outlined in the
Library Construction Protocol: Library Amplification
DNA Input and Fragmentation (step 5).
• The standard SeqCap EZ Library SR workflow specifies
• Use 5 µL of the Library Amplification Primer Mix
100 ng of DNA as the input into library construction
(10X) supplied in the KAPA HyperPlus Kit for each
(the enzymatic fragmentation reaction).
amplification reaction.
Note: libraries may be constructed from lower or higher
• Perform 7 cycles of pre-capture amplification.
inputs, but sequencing metrics are not guaranteed. To
use the KAPA HyperPlus Kit in a SeqCap EZ workflow Note: 7 cycles of pre-capture amplification should yield
with lower or higher DNA inputs, please consult ≥1 µg of pre-capture library, i.e., a sufficient amount to
Important Parameters (pp. 3 – 10) or contact Technical perform individual captures. If you are pooling libraries
Support at [Link]/support. before capture, the amount of DNA needed per
capture is ≥1 µg/n, where n = the number of libraries
• An incubation time in the range of 20 – 30 min at 37°C
per capture. The number of pre-capture amplification
will result in the enzymatic fragmentation of input DNA
cycles may therefore be reduced to achieve the
to an average size of 180 – 220 bp. The optimal time is
lowest possible duplication rates and highest library
somewhat dependent on consumables and equipment
complexity prior to capture.
used, and should be determined empirically. Please
refer to Appendix 2: Optimization of Fragmentation
Cleanup after Pre-capture Library Amplification (LM-PCR)
Time (p. 16) for guidelines on how to optimize
fragmentation time when using the KAPA HyperPlus • Perform a 1X bead-based cleanup of the pre-
Kit in a SeqCap EZ workflow for the first time. capture LM-PCR products, as outlined in the Library
Construction Protocol: Library Amplification (step 6).
Note: Enzymatic fragmentation is sensitive to the
presence of EDTA in DNA samples. Please refer to the Quality Control of Pre-capture Libraries
Important Parameters: Input DNA (p. 4) for guidelines • Please refer to the latest version of the SeqCap EZ
on the fragmentation of EDTA-containing DNA samples. Library SR User’s Guide for more details.
End Repair and A-tailing Target Enrichment
• Follow instructions outlined in the Library Construction • Please refer to Chapters 5 – 8 of the latest version of
Protocol: End Repair and A-tailing (step 2). the SeqCap EZ Library SR User’s Guide to complete
the workflow and generate sequencing-ready, enriched
Adapter Ligation
libraries.
• Follow instructions outlined in the Library Construction
Protocol: Adapter Ligation (step 3).
• Use 5 µL of undiluted SeqCap Indexed Adapter
per reaction.

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

KAPA HyperPlus Kit Technical Data Sheet

For Research Use Only. Not for use in diagnostic procedures. 19

KAPA HyperPlus Kit Technical Data Sheet

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a particular purpose. Kapa Biosystems shall not be liable
for errors or for incidental or consequential damages in
connection with the furnishing, use, or performance of this Note to Purchaser: Limited License
document or of any information contained herein. KAPA HyperPlus Kits are developed, designed and sold
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Kapa Biosystems is not responsible nor will be liable in Users of this product may therefore be required to obtain a
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use of Kapa Biosystems products. Licensed under U.S. Patent nos. 5,338,671 and 5,587,287
Kapa Biosystems does not in any way guarantee or and corresponding patents in other countries.
represent that you will obtain satisfactory results from
using Kapa Biosystems products as described herein. The
only warranties provided to you are included in the Limited
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in connection with your use of Kapa Biosystems products.

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