The library construction process, from enzymatic fragmentation to final library, can be performed in ~ 2 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 after 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.
Conditioning solution not required for the KAPA EvoPlus Workflow. KAPA FragTail Readymix is insensitive to inhibitors.
KAPA Adapters are recommended for use with the KAPA EvoPlus Kits. For assistance with third- party adapter compatibility and ordering, please contact Technical Support for guidelines on the formulation of user-supplied library amplification primers.
KAPA Adapters undergo extensive qPCR- and sequencing-based functional and QC testing to confirm:
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 the 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.
The novel, one-tube KAPA EvoPlus 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.
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.
No, the KAPA Library Amp Primers will be available separately. This is different from our on-market KAPA HyperPrep and HyperPlus Kits. The rationale here is that because the KAPA EvoPlus Kits are optimized for WGS and WES workflows, when combined with our KAPA HyperCap workflow v3.x, the customer will use the truncated KAPA HyperPlex adapters with indexing by PCR (i.e., KAPA Universal Adapter + KAPA UDI Primer Mixes). Also, we know that many customers use their own 3rd party adapters with indexing by PCR.
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.
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.
Upon receipt, immediately store enzymes and reaction buffers at -15℃ to -25℃ 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.