What are the recommended applications for the KAPA HyperPrep Kit for Illumina platforms?
- Whole-genome shotgun sequencing
- Whole-exome or targeting sequencing, using KAPA HyperCap Target Enrichment Probes, Agilent SureSelect, IlluminaTruSeq, or IDT xGen Lockdown Probes, or other hybridization capture systems
- Amplicon sequencing
- Methyl-seq (in combination with the KAPA HiFi Uracil+ Library Amplification ReadyMix)
What are the major steps in library construction?
- End repair and A-tailing, which produces end-repaired, 5′-phosphorylated, 3′-dA-tailed, dsDNA fragments
- Adapter ligation, during which dsDNA adapters with 3′-dTMP overhangs are ligated to 3′-dA-tailed library fragments
- Library amplification (optional), which employs high-fidelity, low-bias PCR to amplify library fragments carrying appropriate adapter sequences on both ends
Are there safe stopping points in the library construction process?
The library construction process, from end repair and A-tailing to final, amplified library, can be performed in less than 3 hours, depending on experience and the number of samples being processed. If necessary, the protocol may be safely paused after completion of the post-ligation cleanup, or prior to post-amplification cleanup.
Purified, adapter-ligated library DNA may be stored at 2°C to 8°C for 1 – 2 weeks, or at -15°C to -25°C for ≤1 month before amplification, target capture and/or sequencing. To avoid degradation, always store DNA in a buffered solution (10 mM Tris-HCl, pH 8.0 – 8.5) when possible, and minimize the number of freeze-thaw cycles.
What are your Covaris shearing recommendations?
- If you are performing a cleanup between shearing and end repair, shear in 10 mM Tris-HCl (pH 8 or 8.5) + 1 mM EDTA
- If you are not performing a cleanup between shearing and end repair, shear in 10 mM Tris-HCl (pH 8 or 8.5) + 0.1 mM EDTA
- Never shear DNA in water
What adapters can I use with this kit and at what concentration?
KAPA Adapters are recommended for use with the KAPA HyperPrep Kits. However, the kit is also compatible with other full-length adapter designs wherein both the sequencing and cluster generation sequences are added during the ligation step, such as those routinely used in KAPA HyperCap and KAPA HyperPETE workflows, TruSeq (Illumina) and SureSelect XT2 (Agilent) kits, and other similar library construction workflows. Custom adapters that are of similar design and are compatible with “TA-ligation” of dsDNA may also be used, remembering that custom adapter designs may impact library construction efficiency.
For assistance with adapter compatibility and ordering, please contact Technical Support for guidelines on the formulation of user-supplied library amplification primers.
Ligation efficiency is robust for adapter-insert molar ratios from 10:1 to >200:1. The recommended adapter concentrations for different inputs are given in the table below. Note that high adapter-insert molar ratios are beneficial for low-input and challenging samples. When optimizing workflows for DNA inputs ≤25 ng, it is recommended that two or three adapter concentrations be evaluated. Try the recommended concentration in the table, as well as one or two additional concentrations in the range that is 2 – 10 times higher.
KAPA Adapters may be used for all inputs with the appropriate dilution. For assistance with adapter compatibility and ordering, please visit sequencing.roche.com/support.
|Adapter stock concentration
|Adapter: insert molar ratio
Where do I find more information about KAPA Adapters?
Please refer to the KAPA Adapter Technical Data Sheets for information about barcode sequences, pooling, kit configurations, formulation, and dilution for different KAPA DNA and RNA library preparation kits and inputs.
What QC testing is performed on KAPA Adapters?
KAPA Adapters undergo extensive qPCR- and sequencing-based functional and QC testing to confirm:
- optimal library construction efficiency
- minimal levels of adapter-dimer formation
- nominal levels of barcode cross-contamination
Library construction efficiency and adapter-dimer formation are assessed in a low-input library construction workflow. The conversion rate achieved in the assay indicates library construction efficiency. This is calculated by measuring the yield of adapter-ligated library (before any amplification) by qPCR (using the KAPA Library Quantification Kit), and expressing this as a % of input DNA. To assess adapter-dimer formation, a modified library construction protocol - designed to measure adapter dimer with high sensitivity - is used.
Barcode cross-contamination is assessed by sequencing. Each adapter is ligated to a unique, synthetic insert of known sequence, using a standard library construction protocol.
How many cycles of library amplification should be used?
If cycled to completion (not recommended) a single 50 µL KAPA HiFi library amplification reaction can produce 8–10 µg of amplified library. To minimize over-amplification and associated undesired artefacts, the number of amplification cycles should be tailored to produce the optimal amount of amplified library required for downstream processes. This is typically in the range of 250 ng – 1.5 µg of final, amplified library.
Quantification of adapter-ligated libraries prior to library amplification can greatly facilitate the optimization of library amplification parameters, particularly when a library construction workflow is first established or optimized. The amount of template DNA (adapter-ligated molecules) available for library amplification may be determined using the KAPA Library Quantification Kit. A simple algorithm can subsequently be used to theoretically predict the number of amplification cycles needed to achieve a specific yield of amplified library. A calculator that can be used for purpose is available on request from support.
The estimated number of amplification cycles for libraries prepared from different amounts of input DNA can be found in the latest version of the KAPA HyperPrep Technical Data Sheet. The actual optimal number of amplification cycles may be 1–3 cycles higher or lower, depending on the sample type and size distribution of the input DNA.
Is library amplification required?
If the amount of adapter-ligated library retrieved after the post-ligation cleanup is sufficient for the next process (e.g. direct sequencing), and the library molecules contain all the adapter motifs needed for that process, library amplification may be omitted. This further streamlines the workflow and reduces the overall library preparation time to <2 hours. The high conversion efficiency achievable with the KAPA HyperPrep Kit enables PCR-free workflows with ≥ 50 ng input DNA. KAPA HyperPrep Kits without amplification reagents (07962339001, 07962355001 and 07962371001) are available for PCR-free workflows.
Is the KAPA Library Amplification Primer Mix compatible with all Illumina libraries?
KAPA HyperPrep Kits contain a highly optimized Library Amplification Primer Mix, designed to eliminate or delay primer depletion during library amplification reactions performed with KAPA HiFi DNA polymerase. The primers in the mix are based on the P5 and P7 Illumina flow cell sequences, and are suitable for the amplification of libraries prepared with full-length adapters. User-supplied primer mixes may be used in combination with incomplete or custom adapters. Please contact us for guidelines on the formulation of user-supplied library amplification primers.
What is the shelf-life and the recommended storage conditions for KAPA HyperPrep Kits?
The enzymes provided in this kit are temperature sensitive, and appropriate care should be taken during shipping and storage. KAPA HyperPrep Kits are shipped on dry ice or ice packs, depending on the country of destination. Upon receipt, immediately store enzymes and reaction buffers at -15°C to -25°C in a constant-temperature freezer. When stored under these conditions and handled correctly, the kit components will retain full activity until the expiry date indicated on the kit label.
How many bead-based clean-ups are required after adapter-ligation?
The novel, one-tube KAPA HyperPrep chemistry leads to less adapter-dimer formation and carry-over. A single bead-based cleanup after adapter ligation is sufficient to remove unused adapter and adapter dimer, even at the high adapter:insert molar ratios recommended for low-input applications. If necessary, a second post-ligation (or size selection step) cleanup may be included to remove all traces of unused adapter and adapter-dimer, especially for PCR-free workflows