• Whole-genome shotgun sequencing
  • Targeted sequencing by solution hybrid selection (i.e. exome or custom capture using the SeqCap EZ, Agilent SureSelect, Illumina TruSeq or IDT xGenLockdown Probes systems)
  • Amplicon sequencing
  • ChIP-seq
  • RNA-seq
  • Methyl-seq (in combination with the KAPA HiFi Uracil+ Library Amplification ReadyMix)

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

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

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

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

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

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

 

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

 

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

 

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

Please refer to the KAPA Single-Indexed and Dual-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

Please refer to the KAPA Single-Indexed and Dual-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.

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.

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

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

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

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

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

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

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

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

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

 

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

 

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

 

 

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

1Input into end repair and A-tailing reaction

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

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

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) is not routinely done, but 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 HyperPrep Kits are shipped on dry ice, depending on the country of destination. Upon receipt, immediately store enzymes and reaction buffers at -20°C in a constant-temperature freezer. When stored under these conditions and handled correctly, the kit components will retain full activity until the expiry date indicated on the kit label.

The novel, one-tube KAPA HyperPrep chemistry leads to less adapter-dimer formation and carry-over. A single bead-based cleanup after adapter ligation is sufficient to remove unused adapter and adapter dimer, even at the high adapter:insert molar ratios recommended for low-input applications. If necessary, a second post-ligation (or size selection step) cleanup may be included to remove all traces of unused adapter and adapter-dimer, especially for PCR-free workflows and/or when dual-indexed adapters are used.