KAPA EvoPrep Kits for pre-fragmented DNA

• Whole-genome shotgun sequencing, using the KAPA HyperCap Evolved workflow v4.x
• Targeted sequencing, using KAPA HyperCap or KAPA HyperPETE workflows

• 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 library, can be performed in ~ 2.3 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 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. To avoid degradation, always store DNA in a buffered solution (10 mM Tris-HCl, pH 8.0 – 8.5) when possible, and minimise 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 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

KAPA Adapters are recommended for use with the KAPA EvoPrep Boost/KAPA EvoPrep 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.

Please refer to the KAPA UDI Adapters and KAPA HyperPlex Adapters 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 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 EvoPrep (Boost) 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.

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 EvoPrep Kit enables PCR-free workflows with ≥ 50 ng input DNA. KAPA EvoPrep Kits without amplification reagents (10153806001, 10153814001, 10153857001 and 10154284001) are available for PCR-free workflows.

No, KAPA Library Amp Primers are available separately. This is different from our on-market KAPA HyperPrep and HyperPlus Kits and designed to support greater flexibility. KAPA EvoPrep Boost/KAPA EvoPrep Kits are optimized for both WGS and WES workflows. For WGS, full-length adapters are typically employed (and are essential for PCR-free workflows). For WES or targeted sequencing with our KAPA HyperCap Evolved workflow v4.x, the truncated KAPA HyperPlex adapters (universal or with UMIs) and indexing by PCR (i.e., using KAPA Universal Adapter + KAPA UDI Primer Mixes) are recommended. We also support customers who prefer to use custom third- party adapters or indexing primers for their application(s).

The KAPA Library Amplification Primer Mix is highly optimized and, 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.

If cycled to completion (not recommended) a single 50 μL KAPA EvoAmp 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.

The readymixes provided in this kit are temperature sensitive, and appropriate care should be taken during shipping and storage. KAPA EvoPrep Kits are shipped on dry ice or ice packs, depending on the country of destination. Upon receipt, immediately store the readymixes 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.