The kit has been validated for library construction from 10 ng – 5 µg of human genomic DNA for whole-genome shotgun sequencing or targeted sequencing. For smaller genomes, or lower complexity samples such as ChIP DNA successful library construction has been achieved from low nanogram to picogram quantities (≥100 pg) of input DNA.
The protocol may be paused safely after any of the bead cleanup steps, as described below:
The proportion of fragmented DNA that is successfully converted to adapter-ligated molecules decreases as input is reduced. When starting library construction (end repair) with >100 ng fragmented DNA, 15%–40% of input DNA is typically recovered as adapter-ligated molecules, whereas the recovery typically ranges from 0.5%–15% for libraries constructed from 100 pg – 100 ng DNA. These figures apply to high quality DNA and can be significantly lower for DNA of lower quality and for workflows that contain additional beads cleanups or size selection prior to library amplification.
KAPA Dual- or Single-Indexed Adapters are recommended for use with KAPA Library Preparation Kits, except for methyl-seq applications. KAPA Library Preparation Kits are also compatible with non-indexed, single-indexed, and dual-indexed adapters that are routinely used in SeqCap EZ, Illumina TruSeq, 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 50:1. As a general guideline, an adapter-insert molar ratio of ~10:1 is recommended for libraries constructed from ≥100 ng of fragmented DNA. Higher adapter-insert molar ratios may be beneficial for low-input and challenging samples.
High Concentration (30 µM) KAPA Single-Indexed Adapter Kits are recommended for library construction from from ≥100 µg inputs, whereas Low Concentration (1.5 µM) KAPA Single-Indexed Adapter Kits are recommended for inputs ≤100 ng. Around 100 ng, the most appropriate concentration depends on library insert size. KAPA Dual-Indexed Adapters may be used for all inputs with the appropriate dilution. For assistance with adapter compatibility and ordering, please visit kapabiosystems.com/support.
Please refer to the KAPA Single- 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:
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.
While a single bead-based cleanup removes most unligated adapter and adapter-dimer, a second bead-based cleanup is recommended to eliminate any remaining adapter species from the library. If size selection is carried out between adapter ligation and library amplification (or clustering), a single post-ligation cleanup with beads (1X) is usually sufficient prior to size selection. If no post-ligation size selection is carried out, two consecutive 1X bead-based cleanups are recommended.
Size selection requirements vary widely according to specific applications. Depending on preference the double-sided size selection procedures presented in the Library Preparation User Guide may be omitted entirely, modified, or replaced with alternative size selection procedures (such as manual agarose gel electrophoresis, excision and purification or an automated DNA size selection (e.g. Sage Science Pippin Prep). Size selection may be carried out at alternative points in the workflow, for example:
KAPA HiFi HotStart is the enzyme provided in the KAPA HiFi HotStart ReadyMix. This is an antibody-based hot-start formulation of KAPA HiFi DNA Polymerase, a novel B-family DNA polymerase engineered for increased processivity and high fidelity.
If cycled to completion, (NOT RECOMMENDED) a single 50 µL library amplification PCR can produce 8 µg – 10 µg (16 ng – 200 ng/µL) of amplified library. To minimize over-amplification and associated undesired artifacts, the number of amplification cycles should be optimized to produce an amplified library with a concentration in the range of 10 ng – 30 ng/µL (0.5 µg – 1.5 µg of PCR product per 50 µL reaction). 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. With the KAPA Library Quantification Kit, the amount of template DNA (adapter-ligated molecules) available for library amplification can be determined accurately. Using a simple algorithm (for exponential amplification), the number of amplification cycles needed to achieve a specific yield of amplified library may be predicted theoretically. The actual optimal number of amplification cycles may be 1–3 cycles higher, particularly for libraries constructed from FFPE DNA or other challenging samples, or libraries with a broad fragment size distribution.
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.
The enzymes in this kit are temperature sensitive and appropriate care should be taken during shipping. Upon receipt, immediately store enzymes and reaction buffers at -20°C in a constant-temperature freezer. The PEG/NaCl Solution may be stored at 2°C to 8°C until the expiry date indicated on the kit label.