• Whole-genome shotgun sequencing
  • Whole-exome or targeting sequencing, using SeqCap®
    EZ, Agilent SureSelect, IlluminaTruSeq, or IDT xGen® Lockdown® Probes, or other hybridization capture systems
  • RNA-Seq (selected applications)

This protocol has been validated for library construction from 1 ng – 1 μg of  double-stranded DNA. Please refer to the table below for recommended inputs of different types of DNA, for different sequencing applications.


Application Sample type Recommended input
WGS Complex gDNA
(high quality)
 50 ng–1 μg
Targt capture
(WES, custom panels)
Complex gDNA
(high quality)
 10 ng–1 μg
WGS, target capture FFPE DNA ≥50
(quality dependent)
WGS Microbial DNA  1 ng–1 μg
WGS (PCR-free) High-quality DNA ≥50 ng (no SS)*
≥500 ng (w/SS)*
Targeted sequencing Long amplicons  ≥1 ng
RNA-seq Full-length/
unfragmented cDNA
 ≥1 ng

*SS=size selection; results in the loss of 60 – 95% of DNA, irrespective of whether a bead- or gel-based technique is used

  • Enzymatic fragmentation, which produces dsDNA fragments
  • End repair and A-Tailing, which produces end-repaired, 5’-phosphorylated, 3’-dA-tailed dsDNA fragments
  • Adapter ligation, during which dsDNA adapters with 3’-dTMP overhangs are ligated to 3’-dA-tailed molecules
  • Library amplification (optional), which employs high-fidelity, low-bias PCR to amplify library fragments carrying appropriate adapters sequences on both ends.

The library construction process, from enzymatic fragmentation to final library, can be performed in 1.5 to 3 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 prior to post-amplification cleanup.

Purified, adapter-ligated library DNA may be stored at 4°C for one week, or at -20°C for at least one month before amplification, target capture, and/or sequencing. Library amplification products may be stored in a similar way, but the post-amplification cleanup should be performed as soon as possible. To avoid degradation, always store DNA in a buffered solution (10 mM Tris-HCl, pH 8.0-8.5) when possible, and minimize the number of freeze-thaw cycles.

KAPA Frag a very complex mixture of different endonucleases and accessory proteins designed to minimize sequence specificity.

The enzymatic fragmentation reaction is very sensitive to the presence of EDTA, which must be removed or neutralized prior to fragmentation. EDTA present in DNA preparations may be removed by performing a column- or bead-based purification or buffer exchange prior to enzymatic fragmentation. This is the recommended strategy, particularly for high-throughput workflows. For optimal fragmentation results, DNA should be resuspended in 10 mM Tris-HCl, pH 8.0-8.5 after pre-fragmentation treatment. Alternatively, the inhibitory effect of the EDTA can be mitigated by the inclusion of KAPA Frag Conditional Solution at the appropriate final concentration in the fragmentation reaction according to the table below:


Final EDTA concentration

in 50 μL rxn

Dilution factor

Volume of

Conditioning Solution (per 100 μL)

Volume of

PCR-grade water (per 100 μL)

0.02 – 0.05 mM 32.0 3.1 μL 96.9 μL
0.1 mM 15.4 6.5 μL 93.5 μL
0.2 mM 7.4 13.5 μL 86.5 μL
0.3 mM 4.8 21.0 μL 79.0 μL
0.4 mM 3.3 30.0 μL 70.0 μL
0.5 mM 2.6 38.8 μL 61.2 μL
0.6 mM 2.2 46.5 μL 53.5 μL
0.7 mM 1.8 56.0 μL 44.0 μL
0.8 mM 1.6 64.0 μL 36.0 μL
0.9 mM 1.4 72.0 μL 28.0 μL
1.0 mM 1.3 80.0 μL 20.0 μL

If you are unsure about the presence or concentration of EDTA in your samples:

  • Perform a column- or bead-based purification or buffer exchange prior to enzymatic fragmentation and resuspend in 10 mM Tris-HCl, pH 8.0-8.5.
  • Set up a series of test reactions with the appropriate amount of input DNA, and different final concentrations of KAPA Frag Conditioning Solution. Please refer to Appendix 2 in the Technical Data Sheet: Optimization of Fragmentation Parameters for more information.

Fragmentation parameter guidelines for high-quality genomic DNA are provided below:


Mode fragement length Incubation time at 37°C* Optimization range
600 bp 5 min 3 – 10 min
350 bp 10 min  5 – 20 min
200 bp 20 min 10 – 25 min
150 bp 30 min 20 – 40 min


*These parameters are a good starting point for high-quality genomic DNA. As the degree of fragmentation (mode size and size distribution of DNA fragments) is controlled by fragmentation time and temperature, both factors may be modulated to achieve the desired results. Please refer to Appendix 2 of the Technical Data Sheet: Optimization of Fragmentation Parameters for more guidelines.

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 (as determined with the KAPA hgDNA Quantification and QC Kit). 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.

Standard fragmentation parameters may result in over-fragmentation of low-complexity samples, such as small viral genomes, plasmids, and long amplicons and cDNA. For these sample types, the fragmentation time may have to be reduced to 5 min or less to achieve the desired mode fragment length. This makes control over the reaction difficult, particularly when a large number of samples are processed manually. To enable more robust and reproducible results, the fragmentation temperature may be decreased (to 30°C or 25°C) to reduce enzymatic activity, thus increasing the time needed to achieve the desired fragment length.

While it is possible to remove aliquots of the fragmentation reaction product for analysis in the integrated fragmentation/library construction workflow, it is most productive to assess the outcome of fragmentation once the entire workflow has been completed for the following reasons:

  • It is difficult and disruptive to process low-volume aliquots in a way that is fully representative of the final library.
  • Fragmentation profiles for low-input samples (1 – 10 ng into fragmentation) may not be informative, even when high sensitivity assays are used.
  • The final size distribution of libraries prepared from FFPE samples is always smaller 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.

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


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


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


High Concentration (30 µM) KAPA Single-Indexed Adapter Kits are recommended for library construction from 10 ng – 1 µg inputs, whereas Low Concentration (1.5 µM) KAPA Single-Indexed Adapter Kits are recommended for inputs <10 ng. KAPA Dual-Indexed Adapters may be used for all inputs with the appropriate dilution. For assistance with adapter compatibility and ordering, please visit kapabiosystems.com/support.

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

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

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

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

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

The proportion of fragmented DNA that is successfully converted to adapter-ligated molecules decreases as input is reduced. When starting library construction with ≥100 ng fragmented DNA, 50 – 100% of input DNA is typically converted to adapter-ligated molecules whereas conversion rates range between 10% to 50% for libraries constructed from ≥10 – 100 ng DNA and 5 – 20% for libraries constructed from 1 – 10 ng. These figures apply to high-quality DNA, and may be lower for DNA of lower quality, e.g., FFPE samples. Workflows with additional bead-based cleanups or size selection prior to adapter ligation are likely to result in lower yield of adapter-ligated molecules.

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 and/or when dual-indexed adapters are used.

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


Size selection should preferably be carried out after the post-ligation cleanup or after library amplification.


The standard KAPA HyperPlus protocol does not include size selection. Detailed protocols for double-sided size selection may be found in Appendix 1 of the Technical Data Sheet.

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


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

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 is shown below:


Amount of adapter-ligated DNA in amplification rxn Number of cycles required to generate 1 μg of library DNA
500 ng 1 – 2
100 ng 3 – 4
50 ng 5 – 6
10 ng 7 – 8
5 ng 8 – 9
1 ng 11 – 12
500 pg 12 – 13

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 Library Preparation Kit enables PCR-free workflows from as little as 50 ng of input DNA. KAPA HyperPlus Library Preparation Kits without amplification reagents (KK8511, KK8513, and KK8515) are available for PCR-free workflows.

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

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

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.

The enzymes provided in this kit are temperature sensitive, and appropriate care should be taken during shipping and storage. KAPA HyperPlus Library Preparation Kits are shipped on dry ice, depending on the country of destination. Upon receipt, immediately store enzymes and reaction buffers.