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KAPA Library Amplification Kits include KAPA HiFi DNA Polymerase, a novel enzyme engineered using our Directed Evolution technology for ultra-high-fidelity and robustness. KAPA HiFi has become the enzyme of choice for next-generation sequencing (NGS) library amplification due to its ability to amplify complex DNA populations with high-fidelity, high-efficiency and very low amplification bias. This results in lower duplication rates and improved coverage of GC- and AT-nch regions, promoters and other challenging regions.
Benefits of KAPA Library Amplification Kits
Higher and more uniform coverage with lower dropout of difficult regions
Higher yields, low duplication rates
Higher success rates with different sample types/applications
Reduced bias and efficient amplification of GC- and AT-rich genomes
Improved representation of all library fragments and sequence regions
Fewer cycles to achieve equivalent yields due to higher amplification efficiency
View data showing broader coverage distribution across the range of GC-content using KAPA HiFi DNA polymerase compared to Phusion DNA Polymerase (Thermo Scientific) or the TruSeq PCR Master Mix (Illumina). See data showing more uniform coverage of GC-rich regions and higher amplification efficiency with the KAPA HiFi Kits compared to NEB Q5.
Figure 1. Coverage depth vs. GC content for indexed Illumina TruSeq libraries. Libraries amplified with KAPA HiFi DNA Polymerase, Phusion DNA Polymerase (Thermo Scientific) or the TruSeq PCR Master Mix (Illumina) were compared to an equivalent unamplified library. Scatter plots of mean sequence coverage depth vs. GC content were generated by analyzing 250 bp windows. GC-rich sequences were under-represented in libraries amplified with Phusion and the TruSeq PCR Master Mix. Library amplification with KAPA HiFi DNA Polymerase resulted in coverage distribution across the range of GC-content that is almost indistinguishable from that of the unamplified control.
Figure 2. Coverage depth vs. GC content for indexed Illumina TruSeq libraries. Libraries amplified with KAPA HiFi DNA Polymerase, Phusion DNA Polymerase (Thermo Scientific) or the TruSeq PCR Master Mix (Illumina) were compared to an equivalent unamplified library. Scatter plots of mean sequence coverage depth vs. GC content were generated by analyzing 250 bp windows. GC-rich sequences were under-represented in libraries amplified with Phusion and the TruSeq PCR Master Mix. Library amplification with KAPA HiFi DNA Polymerase resulted in coverage distribution across the range of GC-content that is almost indistinguishable from that of the unamplified control.
Figure 3. Bias within low GC-content libraries is minimized by KAPA HiFi DNA Polymerase. Libraries prepared from P. falciparum (19% GC), C. difficile (28%), E. coli (51% GC), and B. pertussis (68%) were amplified using KAPA HiFi HotStart ReadyMix or NEB Q5. KAPA HiFi HotStart ReadyMix yielded significantly more even coverage of the C. difficile genome than NEB Q5. As a result much less sequencing is needed to cover all regions to the requisite average coverage depth.
Fig 4. Bias within low GC-content libraries is minimized by KAPA HiFi DNA Polymerase. Libraries prepared from P. falciparum (19% GC), C. difficile (28%), E. coli (51% GC), and B. pertussis (68%) were amplified using KAPA HiFi HotStart ReadyMix or NEB Q5. KAPA HiFi HotStart ReadyMix outperformed NEB Q5 with respect to amplification efficiency across all organisms. Less amplification has a positive impact on key sequencing metrics.
Figure 5. KAPA HiFi HotStart ReadyMix reduces amplification bias and improves sequencing coverage. Libraries were prepared for whole-genome shotgun sequencing from 100 ng of Covaris-sheared human genomic DNA using either the KAPA HTP Library Preparation Kit (first) or a competitor kit (second). Lower amplification bias with KAPA HiFi HotStart ReadyMix resulted in more uniform coverage. Data courtesy of The Broad Institute (Cambridge, MA, USA).
Figure 6. KAPA HiFi HotStart ReadyMix reduces amplification bias and improves sequencing coverage. Libraries were prepared for whole-genome shotgun sequencing from 100 ng of Covaris-sheared human genomic DNA using either the KAPA HTP Library Preparation Kit (left) or a competitor kit (right). Lower amplification bias with KAPA HiFi HotStart ReadyMix resulted in more uniform coverage. Data courtesy of The Broad Institute (Cambridge, MA, USA).
Amplification of NGS libraries with ultra-high fidelity
Enhanced proofreading (3′-5′ exonuclease) activity using KAPA HiFi enzyme
Ultra-high fidelity for NGS applications
View data showing the least amount of error rates during NGC library amplification using KAPA HiFi DNA polymerase compared to Taq, Taq -based blends, Q5 and Phusion.
Figure 7.Industry-leading fidelity confirmed by pyrosequencing. Published error rates of Taq, Taq-based long-range PCR blends, and proofreading DNA polymerases (Q5, New England Biolabs and Phusion, Thermo Scientific) are compared to the error rate of KAPA HiFi HotStart ReadyMix. The error rate of KAPA HiFi HotStart ReadyMix is 50 – 100 times lower than that of Taq. The error rate of KAPA HiFi HotStart ReadyMix was confirmed by deep sequencing on the 454 platform, and is 1 error per 3.54 x 10-6 nucleotides incorporated.
Kits include KAPA HiFi or KAPA HiFi HotStart DNA Polymerase (1 U/µL), Fidelity Buffer (5X), GC Buffer (5X), high-quality dNTPs (10 mM each) and MgCl2 (25 mM). Both buffers contain MgCl2 at a final concentration of 2 mM.
KAPA HiFi HotStart is also available in KAPA HiFi HotStart ReadyMix (2X), which contains DNA Polymerase, reaction buffer, dNTPs and MgCl2 (at a final concentration of 2.5 mM).
