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KAPA HyperPlus Kits

KAPA HyperPlus Kits, our most advanced library preparation option, provide a streamlined workflow that includes fully automatable fragmentation and library preparation in a single tube. This kit is based on our DNA Hyper portfolio which offers  industry-leading library construction efficiencies. This integrated solution combines enzymatic fragmentation, similar in quality to mechanical shearing, with the speed and convenience of tagmentation-based workflows.

KAPA HyperPlus Kits offer a complete library preparation solution with KAPA Adapters and KAPA HyperPure Beads (sold separately). The kits are compatible with the Illumina sequencing platform and provide qualified automation methods.

Features and Benefits of KAPA HyperPlus Kits

• Fully automatable DNA library construction in 1.5–3 hours with low-bias enzymatic fragmentation

• Industry-leading conversion rates and library complexity,* particularly for FFPE DNA

• Now includes an additional End Repair and A-Tailing module for more sensitive applications with no change to the workflow

• PCR-free workflows from lower inputs and improved sequence coverage in workflows with PCR

• Compatibility with a wide range of sample types and inputs, and flexibility with respect to fragment size, adapter design and library amplification 

HyperPlus_Workflow.jpg
Product Highlights

Industry-leading library construction yields* with integrated workflow

  •  Low-input samples no longer require specialist library construction workflows or reagents
  •  PCR-free workflows possible from as little as 50 ng starting material
  •  Both the existing and upgraded KAPA HyperPlus chemistry produce the highest post-ligation yields

Validated with the HyperCap Workflow v2.0

 

Post-ligation yield.png

KAPA HyperPlus Kits produce the highestyields  from a range of input amounts. The KAPA HyperPlus kit (both existing and upgraded workflows) converts more input DNA to adapter-ligated library than NEBNext® Ultra || FS Kits or Covaris shearing combined with KAPA HyperPrep. PCR-free libraries were prepared from 1ng, 10ng and 100ng E.coli gDNA using KAPA UDI Adapters and KAPA HyperPure Beads for all workflows, including NEBNext Ultra || FS Kits. Libraries were quantified after ligation using the KAPA Library Quantification Kit. 

Post-ligation yield.png

KAPA HyperPlus Kits produce the highestyields  from a range of input amounts. The KAPA HyperPlus kit (both existing and upgraded workflows) converts more input DNA to adapter-ligated library than NEBNext® Ultra || FS Kits or Covaris shearing combined with KAPA HyperPrep. PCR-free libraries were prepared from 1ng, 10ng and 100ng E.coli gDNA using KAPA UDI Adapters and KAPA HyperPure Beads for all workflows, including NEBNext Ultra || FS Kits. Libraries were quantified after ligation using the KAPA Library Quantification Kit. 

Tunable and low-bias enzymatic fragmentation

  • Library insert sizes adjustable from 150–800 bp by varying fragmentation time or temperature
  • Robust and reproducible fragmentation across a range of GC content and DNA input amounts and sample types
HyperPlus_Figure-9.jpg

The KAPA fragmentation system enables tunable enzymatic fragmentation.Human genomic DNA (100 ng) was fragmented at 37°C for different periods of time (5 – 45 min) to achieve mode library insert sizes ranging from approximately 150 – 800 bp. The KAPA HyperPlus workflow was completed without any size selection using full-length adapters and 4 cycles of amplification. Final libraries were analyzed using a LabChip GX Touch HT instrument and HT DNA HiSens Reagent Kit (PerkinElmer).

HyperPlus_Figure-5.jpg

Defined fragmentation parameters yield consistent library insert sizes across a range of species and genomic GC content. Genomic DNA (10 ng or 50 ng) from human (41% GC), Bordetella pertussis (68% GC), Clostridium difficile (29% GC), Escherichia coli (51% GC), and Plasmodium falciparum (20% GC) was fragmented at 37ºC for 5, 15, or 45 minutes, yielding average library insert sizes of ~700, ~350 or ~200 bp, respectively. Average fragment sizes for final, amplified libraries were determined with a Bioanalyzer 2100 instrument and High-Sensitivity DNA Assay (Agilent Technologies).

HyperPlus_Figure7a.jpg

The KAPA HyperPlus workflow results in minimal fragmentation bias. Nucleotide content over a 40 bp window (-10 to +30 bp relative to read alignment start) for whole exome libraries were prepared from 50 ng of human gDNA (41% GC) with the KAPA HyperPlus Kit, the KAPA HyperPrep Kit with Covaris shearing, or the Nextera Rapid Capture Exome Kit (Illumina). Enzymatic fragmentation with the KAPA HyperPlus Kit resulted in slightly more start site bias than mechanical shearing, but much less bias than tagmentation. Paired-end sequencing (2 x 100 bp) was performed on an Illumina HiSeq 2500 and analysis performed with a custom python script.

HyperPlus_Figure7b.jpg

The KAPA HyperPlus workflow results in minimal fragmentation bias. Nucleotide content over a 40 bp window (-10 to +30 bp relative to read alignment start) for whole exome libraries were prepared from 50 ng of human gDNA (41% GC) with the KAPA HyperPlus Kit, the KAPA HyperPrep Kit with Covaris shearing, or the Nextera Rapid Capture Exome Kit (Illumina). Enzymatic fragmentation with the KAPA HyperPlus Kit resulted in slightly more start site bias than mechanical shearing, but much less bias than tagmentation. Paired-end sequencing (2 x 100 bp) was performed on an Illumina HiSeq 2500 and analysis performed with a custom python script.

HyperPlus_Figure7c.jpg

The KAPA HyperPlus workflow results in minimal fragmentation bias. Nucleotide content over a 40 bp window (-10 to +30 bp relative to read alignment start) for whole exome libraries were prepared from 50 ng of human gDNA (41% GC) with the KAPA HyperPlus Kit, the KAPA HyperPrep Kit with Covaris shearing, or the Nextera Rapid Capture Exome Kit (Illumina). Enzymatic fragmentation with the KAPA HyperPlus Kit resulted in slightly more start site bias than mechanical shearing, but much less bias than tagmentation. Paired-end sequencing (2 x 100 bp) was performed on an Illumina HiSeq 2500 and analysis performed with a custom python script.

HyperPlus_Figure-9.jpg

The KAPA fragmentation system enables tunable enzymatic fragmentation.Human genomic DNA (100 ng) was fragmented at 37°C for different periods of time (5 – 45 min) to achieve mode library insert sizes ranging from approximately 150 – 800 bp. The KAPA HyperPlus workflow was completed without any size selection using full-length adapters and 4 cycles of amplification. Final libraries were analyzed using a LabChip GX Touch HT instrument and HT DNA HiSens Reagent Kit (PerkinElmer).

HyperPlus_Figure-5.jpg

Defined fragmentation parameters yield consistent library insert sizes across a range of species and genomic GC content. Genomic DNA (10 ng or 50 ng) from human (41% GC), Bordetella pertussis (68% GC), Clostridium difficile (29% GC), Escherichia coli (51% GC), and Plasmodium falciparum (20% GC) was fragmented at 37ºC for 5, 15, or 45 minutes, yielding average library insert sizes of ~700, ~350 or ~200 bp, respectively. Average fragment sizes for final, amplified libraries were determined with a Bioanalyzer 2100 instrument and High-Sensitivity DNA Assay (Agilent Technologies).

HyperPlus_Figure7a.jpg

The KAPA HyperPlus workflow results in minimal fragmentation bias. Nucleotide content over a 40 bp window (-10 to +30 bp relative to read alignment start) for whole exome libraries were prepared from 50 ng of human gDNA (41% GC) with the KAPA HyperPlus Kit, the KAPA HyperPrep Kit with Covaris shearing, or the Nextera Rapid Capture Exome Kit (Illumina). Enzymatic fragmentation with the KAPA HyperPlus Kit resulted in slightly more start site bias than mechanical shearing, but much less bias than tagmentation. Paired-end sequencing (2 x 100 bp) was performed on an Illumina HiSeq 2500 and analysis performed with a custom python script.

HyperPlus_Figure7b.jpg

The KAPA HyperPlus workflow results in minimal fragmentation bias. Nucleotide content over a 40 bp window (-10 to +30 bp relative to read alignment start) for whole exome libraries were prepared from 50 ng of human gDNA (41% GC) with the KAPA HyperPlus Kit, the KAPA HyperPrep Kit with Covaris shearing, or the Nextera Rapid Capture Exome Kit (Illumina). Enzymatic fragmentation with the KAPA HyperPlus Kit resulted in slightly more start site bias than mechanical shearing, but much less bias than tagmentation. Paired-end sequencing (2 x 100 bp) was performed on an Illumina HiSeq 2500 and analysis performed with a custom python script.

HyperPlus_Figure7c.jpg

The KAPA HyperPlus workflow results in minimal fragmentation bias. Nucleotide content over a 40 bp window (-10 to +30 bp relative to read alignment start) for whole exome libraries were prepared from 50 ng of human gDNA (41% GC) with the KAPA HyperPlus Kit, the KAPA HyperPrep Kit with Covaris shearing, or the Nextera Rapid Capture Exome Kit (Illumina). Enzymatic fragmentation with the KAPA HyperPlus Kit resulted in slightly more start site bias than mechanical shearing, but much less bias than tagmentation. Paired-end sequencing (2 x 100 bp) was performed on an Illumina HiSeq 2500 and analysis performed with a custom python script.

Superior sequencing results with KAPA HyperPlus Kits

  • Higher post-ligation yields result in fewer amplification cycles and lower duplication rates
  •  Detect low-frequency mutations with high confidence due to decreased sequencing artifacts and increased sensitivity with the upgraded version of the KAPA HyperPlus Kit
Duplication Rates.png

Lower duplication rates. Whole human exome libraries were prepared using 100 ng inputs with the existing and upgraded KAPA HyperPlus Kit. Captures were performed with the SeqCap EZ MedExome panel. 

Sequencing_quality620x400.png

Improved sequencing performance. Whole human exome libraries were prepared using 100 ng inputs with the existing and upgraded KAPA HyperPlus Kit. Captures were performed with the SeqCap EZ MedExome panel. Strand-split artifact reads (SSARs) represent chimeric reads that appear to be derived from non-contiguous portions of the genome1. 1 Source: Haile, et al. (2019) Sources of erroneous sequences and artifact chimeric reads in next generation sequencing of genomic DNA from formalin-fixed paraffin-embedded samples. Nucleic Acids Research, 2019, 47,2. doi: 10.1093/nar/gky1142.

Sensitivity.png

Higher single nucleotide polymorphism (SNP) sensitivity. Whole human exome libraries were prepared using 100 ng high-quality gDNA with the KAPA HyperPrep Kit, the existing and upgraded KAPA HyperPlus Kit. Captures were performed with the SeqCap EZ MedExome panel.

Duplication Rates.png

Lower duplication rates. Whole human exome libraries were prepared using 100 ng inputs with the existing and upgraded KAPA HyperPlus Kit. Captures were performed with the SeqCap EZ MedExome panel. 

Sequencing_quality620x400.png

Improved sequencing performance. Whole human exome libraries were prepared using 100 ng inputs with the existing and upgraded KAPA HyperPlus Kit. Captures were performed with the SeqCap EZ MedExome panel. Strand-split artifact reads (SSARs) represent chimeric reads that appear to be derived from non-contiguous portions of the genome1. 1 Source: Haile, et al. (2019) Sources of erroneous sequences and artifact chimeric reads in next generation sequencing of genomic DNA from formalin-fixed paraffin-embedded samples. Nucleic Acids Research, 2019, 47,2. doi: 10.1093/nar/gky1142.

Sensitivity.png

Higher single nucleotide polymorphism (SNP) sensitivity. Whole human exome libraries were prepared using 100 ng high-quality gDNA with the KAPA HyperPrep Kit, the existing and upgraded KAPA HyperPlus Kit. Captures were performed with the SeqCap EZ MedExome panel.

Improved coverage depth for FFPE samples compared to Covaris-sheared DNA

  • Significant increase in coverage depth compared to KAPA HyperPrep Kits using Covaris-sheared DNA
  • Lower duplication rates, more on-target reads and more bases covered compared to KAPA HyperPrep Kits using Covaris-sheared DNA
  • Detection of variants with high degree of correlation between libraries prepared from KAPA HyperPrep Kits using  Covaris-sheared DNA versus HyperPlus Kits*
HyperPlus_Figure8a.jpg

KAPA HyperPlus Kits enable improved coverage for FFPE samples. Libraries were prepared from 200 ng of FFPE DNA or 50 ng of matched normal controls (DNA from blood) using the KAPA HyperPrep Kit with Covaris® shearing, or the KAPA HyperPlus Kit. Two rounds of multiplexed target capture were performed with a custom SeqCap® EZ panel targeting regions of 42 genes associated with gastrointestinal cancer. Significantly lower duplication rates for the KAPA HyperPlus libraries and slightly higher on-target rates translated to a significant increase in coverage depth for the FFPE samples. Variants at an expected frequency ≥5% were detected with a high degree of correlation (R2 >0.96) between the libraries prepared from Covaris-sheared vs. enzymatically fragmented DNA. Paired-end sequencing (2 x 150 bp) was performed on an Illumina MiSeq and data analyzed with Picard and Illumina VariantStudio v2.2. Data courtesy of The Royal Marsden Hospital.

HyperPlus_Figure8b.jpg

KAPA HyperPlus Kits enable improved coverage for FFPE samples. Libraries were prepared from 200 ng of FFPE DNA or 50 ng of matched normal controls (DNA from blood) using the KAPA HyperPrep Kit with Covaris® shearing, or the KAPA HyperPlus Kit. Two rounds of multiplexed target capture were performed with a custom SeqCap® EZ panel targeting regions of 42 genes associated with gastrointestinal cancer. Significantly lower duplication rates for the KAPA HyperPlus libraries and slightly higher on-target rates translated to a significant increase in coverage depth for the FFPE samples. Variants at an expected frequency ≥5% were detected with a high degree of correlation (R2 >0.96) between the libraries prepared from Covaris-sheared vs. enzymatically fragmented DNA. Paired-end sequencing (2 x 150 bp) was performed on an Illumina MiSeq and data analyzed with Picard and Illumina VariantStudio v2.2. Data courtesy of The Royal Marsden Hospital.

HyperPlus_Figure8c.jpg

KAPA HyperPlus Kits enable improved coverage for FFPE samples. Libraries were prepared from 200 ng of FFPE DNA or 50 ng of matched normal controls (DNA from blood) using the KAPA HyperPrep Kit with Covaris® shearing, or the KAPA HyperPlus Kit. Two rounds of multiplexed target capture were performed with a custom SeqCap® EZ panel targeting regions of 42 genes associated with gastrointestinal cancer. Significantly lower duplication rates for the KAPA HyperPlus libraries and slightly higher on-target rates translated to a significant increase in coverage depth for the FFPE samples. Variants at an expected frequency ≥5% were detected with a high degree of correlation (R2 >0.96) between the libraries prepared from Covaris-sheared vs. enzymatically fragmented DNA. Paired-end sequencing (2 x 150 bp) was performed on an Illumina MiSeq and data analyzed with Picard and Illumina VariantStudio v2.2. Data courtesy of The Royal Marsden Hospital.

HyperPlus_Figure8d.jpg

KAPA HyperPlus Kits enable improved coverage for FFPE samples. Libraries were prepared from 200 ng of FFPE DNA or 50 ng of matched normal controls (DNA from blood) using the KAPA HyperPrep Kit with Covaris® shearing, or the KAPA HyperPlus Kit. Two rounds of multiplexed target capture were performed with a custom SeqCap® EZ panel targeting regions of 42 genes associated with gastrointestinal cancer. Significantly lower duplication rates for the KAPA HyperPlus libraries and slightly higher on-target rates translated to a significant increase in coverage depth for the FFPE samples. Variants at an expected frequency ≥5% were detected with a high degree of correlation (R2 >0.96) between the libraries prepared from Covaris-sheared vs. enzymatically fragmented DNA. Paired-end sequencing (2 x 150 bp) was performed on an Illumina MiSeq and data analyzed with Picard and Illumina VariantStudio v2.2. Data courtesy of The Royal Marsden Hospital.

HyperPlus_Figure8a.jpg

KAPA HyperPlus Kits enable improved coverage for FFPE samples. Libraries were prepared from 200 ng of FFPE DNA or 50 ng of matched normal controls (DNA from blood) using the KAPA HyperPrep Kit with Covaris® shearing, or the KAPA HyperPlus Kit. Two rounds of multiplexed target capture were performed with a custom SeqCap® EZ panel targeting regions of 42 genes associated with gastrointestinal cancer. Significantly lower duplication rates for the KAPA HyperPlus libraries and slightly higher on-target rates translated to a significant increase in coverage depth for the FFPE samples. Variants at an expected frequency ≥5% were detected with a high degree of correlation (R2 >0.96) between the libraries prepared from Covaris-sheared vs. enzymatically fragmented DNA. Paired-end sequencing (2 x 150 bp) was performed on an Illumina MiSeq and data analyzed with Picard and Illumina VariantStudio v2.2. Data courtesy of The Royal Marsden Hospital.

HyperPlus_Figure8b.jpg

KAPA HyperPlus Kits enable improved coverage for FFPE samples. Libraries were prepared from 200 ng of FFPE DNA or 50 ng of matched normal controls (DNA from blood) using the KAPA HyperPrep Kit with Covaris® shearing, or the KAPA HyperPlus Kit. Two rounds of multiplexed target capture were performed with a custom SeqCap® EZ panel targeting regions of 42 genes associated with gastrointestinal cancer. Significantly lower duplication rates for the KAPA HyperPlus libraries and slightly higher on-target rates translated to a significant increase in coverage depth for the FFPE samples. Variants at an expected frequency ≥5% were detected with a high degree of correlation (R2 >0.96) between the libraries prepared from Covaris-sheared vs. enzymatically fragmented DNA. Paired-end sequencing (2 x 150 bp) was performed on an Illumina MiSeq and data analyzed with Picard and Illumina VariantStudio v2.2. Data courtesy of The Royal Marsden Hospital.

HyperPlus_Figure8c.jpg

KAPA HyperPlus Kits enable improved coverage for FFPE samples. Libraries were prepared from 200 ng of FFPE DNA or 50 ng of matched normal controls (DNA from blood) using the KAPA HyperPrep Kit with Covaris® shearing, or the KAPA HyperPlus Kit. Two rounds of multiplexed target capture were performed with a custom SeqCap® EZ panel targeting regions of 42 genes associated with gastrointestinal cancer. Significantly lower duplication rates for the KAPA HyperPlus libraries and slightly higher on-target rates translated to a significant increase in coverage depth for the FFPE samples. Variants at an expected frequency ≥5% were detected with a high degree of correlation (R2 >0.96) between the libraries prepared from Covaris-sheared vs. enzymatically fragmented DNA. Paired-end sequencing (2 x 150 bp) was performed on an Illumina MiSeq and data analyzed with Picard and Illumina VariantStudio v2.2. Data courtesy of The Royal Marsden Hospital.

HyperPlus_Figure8d.jpg

KAPA HyperPlus Kits enable improved coverage for FFPE samples. Libraries were prepared from 200 ng of FFPE DNA or 50 ng of matched normal controls (DNA from blood) using the KAPA HyperPrep Kit with Covaris® shearing, or the KAPA HyperPlus Kit. Two rounds of multiplexed target capture were performed with a custom SeqCap® EZ panel targeting regions of 42 genes associated with gastrointestinal cancer. Significantly lower duplication rates for the KAPA HyperPlus libraries and slightly higher on-target rates translated to a significant increase in coverage depth for the FFPE samples. Variants at an expected frequency ≥5% were detected with a high degree of correlation (R2 >0.96) between the libraries prepared from Covaris-sheared vs. enzymatically fragmented DNA. Paired-end sequencing (2 x 150 bp) was performed on an Illumina MiSeq and data analyzed with Picard and Illumina VariantStudio v2.2. Data courtesy of The Royal Marsden Hospital.

Minimal sequence coverage bias
  • Lower sequence bias when compared other enzymatic fragmentation methods
  • Less bias leads to more uniform sequencing coverage and reduced sequencing costs
GC-bias-comparison-C.difficile.png

GC bias comparision. GC bias for C. difficile, E. coli and B. pertussis was assessed by calculating the GC content of the reference in 100 bp and bins and plotting normalized coverage across these bins for the KAPA HyperPlus, NEBNext® Ultra™ FS and QIAseq FX workflows, using Picard CollectGCBiasMetrics. Libraries were prepared from 10 ng of input DNA. In the absence of sequencing bias, all bins would be equally represented, indicated by a horizontal distribution centered on a normalized coverage of 1. Distribution of GC content in the genome is indicated by the grey histograms.

GC-bias-comparison-Ecoli.png

GC bias comparision. GC bias for C. difficile, E. coli and B. pertussis was assessed by calculating the GC content of the reference in 100 bp and bins and plotting normalized coverage across these bins for the KAPA HyperPlus, NEBNext® Ultra™ FS and QIAseq FX workflows, using Picard CollectGCBiasMetrics. Libraries were prepared from 10 ng of input DNA. In the absence of sequencing bias, all bins would be equally represented, indicated by a horizontal distribution centered on a normalized coverage of 1. Distribution of GC content in the genome is indicated by the grey histograms.

GC-bias-comparison-B.pertussis.png

GC bias comparision. GC bias for C. difficile, E. coli and B. pertussis was assessed by calculating the GC content of the reference in 100 bp and bins and plotting normalized coverage across these bins for the KAPA HyperPlus, NEBNext® Ultra™ FS and QIAseq FX workflows, using Picard CollectGCBiasMetrics. Libraries were prepared from 10 ng of input DNA. In the absence of sequencing bias, all bins would be equally represented, indicated by a horizontal distribution centered on a normalized coverage of 1. Distribution of GC content in the genome is indicated by the grey histograms.

GC-bias-comparison-C.difficile.png

GC bias comparision. GC bias for C. difficile, E. coli and B. pertussis was assessed by calculating the GC content of the reference in 100 bp and bins and plotting normalized coverage across these bins for the KAPA HyperPlus, NEBNext® Ultra™ FS and QIAseq FX workflows, using Picard CollectGCBiasMetrics. Libraries were prepared from 10 ng of input DNA. In the absence of sequencing bias, all bins would be equally represented, indicated by a horizontal distribution centered on a normalized coverage of 1. Distribution of GC content in the genome is indicated by the grey histograms.

GC-bias-comparison-Ecoli.png

GC bias comparision. GC bias for C. difficile, E. coli and B. pertussis was assessed by calculating the GC content of the reference in 100 bp and bins and plotting normalized coverage across these bins for the KAPA HyperPlus, NEBNext® Ultra™ FS and QIAseq FX workflows, using Picard CollectGCBiasMetrics. Libraries were prepared from 10 ng of input DNA. In the absence of sequencing bias, all bins would be equally represented, indicated by a horizontal distribution centered on a normalized coverage of 1. Distribution of GC content in the genome is indicated by the grey histograms.

GC-bias-comparison-B.pertussis.png

GC bias comparision. GC bias for C. difficile, E. coli and B. pertussis was assessed by calculating the GC content of the reference in 100 bp and bins and plotting normalized coverage across these bins for the KAPA HyperPlus, NEBNext® Ultra™ FS and QIAseq FX workflows, using Picard CollectGCBiasMetrics. Libraries were prepared from 10 ng of input DNA. In the absence of sequencing bias, all bins would be equally represented, indicated by a horizontal distribution centered on a normalized coverage of 1. Distribution of GC content in the genome is indicated by the grey histograms.

Featured Webinar

NGS library preparation: Theory and practice
Part 3: A practical example—Library preparation with the KAPA HyperPlus Kit

Presenter: Dr. Maryke Appel, Roche

*Data on file.

Research Use Only. Not for use in diagnostic procedures.
KAPA is a trademark of Roche. All other product names and trademarks are the property of their respective owners.

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Specifications
Spec
 Description
Compatible Platform
 All Illumina sequencing instruments
Library Type
 DNA
Starting Material
 genomic DNA, FFPE DNA, PCR amplicons
Input Amount
 1 ng – 1 µg
Kit storage -15°C to -25°C

Research Use Only. Not for use in diagnostic procedures.
KAPA is a trademark of Roche. All other product names and trademarks are the property of their respective owners.


Technical Documents

Instructions for product use and other product-related technical documents

Research Use Only. Not for use in diagnostic procedures.
KAPA is a trademark of Roche. All other product names and trademarks are the property of their respective owners.

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Kit Code Roche Cat. No Description Kit Size (reactions) How to buy
KK8510 07962380001 KAPA HyperPlus Kit 8 Contact Us
KK8512 07962401001 KAPA HyperPlus Kit 24 Contact Us
KK8514 07962428001 KAPA HyperPlus Kit 96 Contact Us
KK8511 07962398001 KAPA HyperPlus Kit (PCR-free) 8 Contact Us
KK8513 07962410001 KAPA HyperPlus Kit (PCR-free) 24 Contact Us
KK8515 07962436001 KAPA HyperPlus Kit (PCR-free) 96 Contact Us

Accessory Products

All KAPA Adapter Kits contain KAPA Adapter Dilution Buffer and three additional sealing films to support multiple use. KAPA Adapter Dilution Buffer (08278539001) is also available separately, if required.

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Kit Code Roche Cat. No Description Kit Size How to buy
KK8007 08963835001 KAPA HyperPure Beads (5 mL) 5 mL Contact Us
KK8008 08963843001 KAPA HyperPure Beads (30 mL) 30 mL Contact Us
KK8009 08963851001 KAPA HyperPure Beads (60 mL) 60 mL Contact Us
KK8011 08963878001 KAPA HyperPure Beads (4 x 60 mL) 4 x 60 mL Contact US
KK8010 08963860001 KAPA HyperPure Beads (450 mL) 450 mL Contact Us
KK8727 08861919702 KAPA Unique Dual-Indexed Adapters Kit (15μM) 96 adapters x 20 μL each Contact Us
KK8722 08278555702 KAPA Dual-Indexed Adapter Kit (15μM) 96 adapters x 20 μL each Contact Us
KK8721 08278539001 KAPA Adapter Dilution Buffer (25 mL) 25mL Contact Us

Additional pack sizes are available for KAPA HyperPure Beads. Please speak to your local sales representative.

Research Use Only. Not for use in diagnostic procedures.
KAPA is a trademark of Roche. All other product names and trademarks are the property of their respective owners.

  • Whole-genome shotgun sequencing
  • Whole-exome or targeting sequencing, using SeqCap®
    EZ, Agilent SureSelect, IlluminaTruSeq, or IDT xGen® Lockdown® Probes, or other hybridization capture systems
  • RNA-Seq (selected applications)

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. 

Input DNA

 Adapter stock concentration

Adapter:insert molar ratio

  
1 μg
 15 μM
 10:1  
500ng 15 μM
 20:1  
250ng 15 μM
 40:1  
100ng 15 μM
 100:1  
50ng 15 μM
 200:1  
25ng 7.5 μM

 200:1  
10 ng 3 μM
 200:1  
5 ng 1.5 μM
 200:1  
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. 

Research Use Only. Not for use in diagnostic procedures.
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