KAPA EvoAmp ReadyMix powered by HiFi

The KAPA EvoAmp ReadyMix is ideally suited for the amplification of NGS libraries carrying appropriate adapter sequences and the enrichment of targeted capture sequences where high efficiency, low-bias and high-fidelity amplification is required.

No, the KAPA Library Amp Primers will be available separately. This is different from our on-market KAPA Library Amplification Kits. The rationale here is that depending on the application of choice, customers will use either full length (i.e., KAPA UDI Adapters) for WGS or truncated adapters (i.e., KAPA Universal Adapter + KAPA UDI Primer Mixes) for target sequencing workflows. 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 EvoAmp ReadyMix 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.

These higher molecular weight peaks are artifacts of over-amplification. In library amplification reactions, primers are typically depleted before dNTPs. When DNA synthesis can no longer take place due to primer depletion, subsequent rounds of DNA denaturation and annealing result in the separation of complementary DNA strands, followed by imperfect annealing to non-complementary partners by way of the adapter sequences. This presumably results in the formation of long, mostly single-stranded, so-called “daisy-chains”, comprising large assemblies of improperly annealed, partially double-stranded, heteroduplex DNA.

In most cases the “daisy-chained” molecules are bona fide library molecules that are temporarily annealed to one another to form longer concatemers. Since these heteroduplexes contain significant portions of single-stranded DNA, over-amplification leads to the under-quantification of library molecules with assays employing dsDNA-binding dyes. qPCR-based library quantification methods, such as the KAPA Library Quantification assay, quantify DNA by denaturation and amplification, thereby providing an accurate measure of the amount of adapter-ligated molecules in a library, even if the library was over-amplified.

The optimal number of amplification cycles may be 1 – 3 cycles higher for libraries constructed from FFPE DNA or other challenging samples, or libraries with a broad fragment size distribution.

Excessive library amplification can result in unwanted artifacts such as PCR duplicates, chimeric library inserts, and nucleotide substitutions. The extent of library amplification should therefore be limited as much as possible, while ensuring that sufficient material is generated for QC and downstream processing (sequencing or target enrichment).

The KAPA EvoAmp ReadyMix is temperature sensitive, and appropriate care should be taken during shipping and storage. The KAPA EvoAmp ReadyMix is shipped on dry ice or ice packs, depending on the country of destination. Upon receipt, immediately store the KAPA EvoAmp ReadyMix at -15°C to -25°C in a constant-temperature freezer. When stored under these conditions and handled correctly, the KAPA EvoAmp ReadyMix will retain full activity until the expiry date indicated on the kit label.