The library construction process, from enzymatic fragmentation to final library, can be performed in I .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 2°C to 8°C for 1 — 2 weeks, or at -15°C to -25°C for S1 month before amplification, target capture and/or sequencing. To avoid degradation, always store DNA in a buffered solution (10 mM Tris-HCI, pH 8.0 — 8.5) when possible, and minimize the number of freeze-thaw cycles.
Two workflows are available for the End Repair and A-tailing step. This protocol has been validated for use with either the HyperPrep End Repair & A-Tailing Enzyme Mix (existing chemistry) or the HyperPlus End Repair & A-Tailing Enzyme Mix (enhanced chemistry). The formulation of the HyperPlus End Repair & A-Tailing Enzyme Mix has been enhanced to improve performance for more sensitive assays. There are no changes to the KAPA HyperPlus workflow, except the option exists to use either of the End Repair & A-Tailing Enzyme Mixes during the End Repair and A-tailing step.
For all new applications, it is recommended to use the HyperPlus End Repair & A-Tailing Enzyme Mix (enhanced chemistry). For existing validated workflows, it is recommended to perform a side-by-side comparison using the two enzyme mixes and determine which one is most suitable for the specific
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.
KAPA Adapters are recommended for use with the KAPA HyperPlus 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 SeqCap EZ, 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 at www.sequencing.roche.com/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 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 s25 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 www.sequencing.roche.com/support.
|Adapter stock concentration||
Adapter:insert molar ratio
|10 ng||3 μM
|5 ng||1.5 μM
|2.5 ng||750 nM||200:1|
|1 ng||300 nM||200:1|
Please refer to the KAPA 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 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 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
If cycled to completion (not recommended) a single 50 PL KAPA HiFi library amplification reaction can produce 8 — 10 pg 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 pg 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 pg of amplified library DNA from adapter-ligated library DNA is shown below:
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 Sl .5 hours. The high conversion efficiency achievable with the KAPA HyperPlus Kit enables PCR-free workflows from as little as 50 ng of input DNA. KAPA HyperPlus Kits without amplification reagents (07962398001, 07962410001 and 07962436001) are available for PCR-free workflows
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 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 -25zc 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.