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KAPA Library Amplification Kits

For Research Use Only. Not for use in diagnostic procedures.
KAPA Library Amplification Kit

Overview

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

Resources

Technical documents

Product Highlights

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.

 

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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 1. Coverage depth vs. GC content

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 2. Coverage Depth

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.

Figure 3. Bias within low GC-content

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.

Fig 4. Bias within low GC-content
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Improved sequencing coverage

  • Improved coverage uniformity of GC- and AT-rich regions, promoters, and other challenging regions
  • Increased coverage depth of difficult regions in targeted capture workflows, where two amplification steps are performed

View comparative data on more uniform coverage across the GC windows using KAPA HiFi enzyme compared to a competitor kit.

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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 5

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).

Figure 6. KAPA HiFi HotStart ReadyMix
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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.

Industry-leading fidelity confirmed by pyrosequencing

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.

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*Data on file.

Store kits for 18 months at -20°C.

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).

Specifications

  • Compatible Platform

    Illumina HiSeq, MiSeq, NextSeq, and GAIIx

  • Library Prep

    DNA, RNA, and bisulfite-treated DNA

  • Starting Material

    Any NGS library that requires amplification

  • Sequencing Applications

    Whole Genome Sequencing, Whole Exome Sequencing,  Targeted Sequencing (custom panels), RNA-Seq, ChIP-Seq, Amplicon Sequencing

Components

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Components

Spec 2XKAPA HiFi HotStart ReadyMix 2X KAPA HiFI HotStart Real time PCR Master Mix Fluorescein Primers
KAPA HiFi HotStart Library Amp Kit X      
       
KAPA HiFi HotStart Library Amp Kit with Primer Mix X     X
       
KAPA HiFi HotStart Real-time Library Amp kit   X X  
       
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KAPA Code
 Roche Cat. No
 Description
 Kit Size
 How to buy
KK2611
 07958951001
 KAPA HiFi HotStart Library Amp Kit 50 x 50 µL reactions
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KK2612
 07958960001
 KAPA HiFi HotStart Library Amp Kit 250 x 50 µL reactions
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KK2620
 07958978001
 KAPA HiFi HotStart Library Amp Kit with Primer Mix 50 x 50 µL reactions
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KK2621
 07958986001
 KAPA HiFi HotStart Library Amp Kit with Primer Mix 250 x 50 µL reactions
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KK2702
 07959028001
 KAPA HiFi HotStart Real-Time Library Amp Kit 250 x 50 µL reactions
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KK2623
 07958994001
 KAPA Library Amp Primer Mix 250 reactions
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09420410001
 09420410001

KAPA Library Amp Primer Mix (384 rxn)

 384 rxn
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09420479001
 09420479001

KAPA Library Amp Primer Mix, 96-well plate

 96-well plate
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