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KAPA EvoPrep Kits

For Research Use Only. Not for use in diagnostic procedures.

KAPA EvoPrep Kits

Overview


The KAPA EvoPrep Kits represent Roche's latest advancements in high-performance, streamlined, and automation-friendly library preparation research solutions. These kits have been validated using a variety of challenging sample inputs, including cell-free DNA, FFPET DNA, and mechanically sheared DNA, and are designed to streamline and reduce workflow steps. The kits feature ReadyMix reagents, available in both tube and plate formats, to facilitate automation. The KAPA EvoT4 DNA Ligase within the ligation ReadyMix of the kits is an evolved potent ligase that is specifically engineered to increase library conversion rates.

 

Features and benefits of KAPA EvoPrep Kits*

Improved sensitivity, specificity, and ligation efficiency with the addition of our new evolved KAPA EvoT4 DNA ligase, in the kit’s ligation ReadyMix.

  • Excellent sequencing performance to support both germline and somatic research applications from challenging sample types such as cfDNA*
  • Ligation time of just 5 minutes, shortening workflow duration without compromising results
  • Analyze more samples with a wide range of DNA inputs (0.1 ng - 500 ng)
  • PCR-free workflows starting from 50 ng of DNA input
  • Capture more unique input molecules with high yields and high conversion rates of up to 100%**
  • Streamlined and convenient workflows with REACH-compliant*** reagents
  • Enhanced flexibility with an 18-month shelf life and better automatability with stabilized ReadyMixes (up to 20 freeze-thaw cycles)
  • Validated with the KAPA HyperCap and KAPA HyperPETE portfolios to enable accelerated target enrichment workflows


* Comparisons are to the KAPA HyperPrep Kit. Data on file
** Dependent on input and sample type
*** Registration, Evaluation, Authorisation and Restriction of Chemicals

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Product highlights

Simplified and streamlined workflows
  • Reduces workflow time with fewer reagents (ReadyMix formulations) and minimal manual steps
  • Tubes and plated format, offer increased efficiency and convenience
  • Manual- and automation-friendly protocol
  • Validated with KAPA HyperCap and KAPA HyperPETE Workflows for accelerated target enrichment

Figure 1: The KAPA EvoPrep Workflow

The KAPA EvoPrep Kit simplifies and streamlines sample prep workflows to reduce complexities, risk for errors, and turnaround times.

*KAPA UDI Adapter Kit and KAPA Library Amplification Primer Mixes, KAPA Universal (UMI) Adapter and KAPA UDI Primer Mixes, and KAPA Cleanup Beads sold separately

The KAPA EvoPrep Kit simplifies and streamlines sample prep workflows to reduce complexities, risk for errors, and turnaround times
Exceptional library yields and sequencing quality
  • Achieve higher library yields across a range of input DNA and sample type
  • Fewer amplification cycles for downstream processing result in lower duplication rates and higher sequence coverage
  • Achieve successful library construction with clinically relevant samples and PCR-free workflows (from as little as 50 ng)

Figure 2: KAPA EvoPrep chemistry enables high library conversion across a range of input DNA

0.1 ng - 500 ng of Covaris-sheared human genomic DNA was used to prepare libraries with KAPA Universal Adapters (with KAPA UDI Primer Mixes) at the recommended adapter: insert molar ratio following the KAPA EvoPrep and KAPA HyperPrep Kit Instructions for Use. Libraries were amplified for various cycles dependent on DNA input to enable visualization. Electropherograms were generated with LabChip GX Touch NGS 3K Assay.

*Non-validated input (outside of the input range) for KAPA HyperPrep Kit - optimized cycle number for KAPA EvoPrep Kit inputs used

KAPA EvoPrep chemistry enables high library conversion across a range of input DNA
Optimal utilization of sequencing throughput
  • Minimal sequence coverage bias leading to more uniform sequencing coverage and reduced sequencing cost
  • Improved sequencing metrics, allowing higher confidence in data due to reduction in sequencing artefacts

Figure 3: Improved sequencing performance - reduction in Chimeras

PCR-free whole genome libraries were prepared using 100 ng of Covaris-sheared human genomic DNA (NA12878) with the KAPA EvoPrep Kit, Supplier I, Supplier Q and Supplier W, following each supplier’s instructions for use. The KAPA EvoPrep Kit had the lowest percentage of Chimeras present, resulting in higher data confidence* compared to Supplier I, Supplier Q and Supplier W having a higher percentage of Chimeras present. 

*Source: Chen, et al. (2024) Characterization and mitigation of artifacts derived from NGS library preparation due to structure-specific sequences in the human genome. BMC Genomics (2024) 25:227 https://doi.org/10.1186/s12864-024-10157-w

Improved sequencing performance - reduction in Chimeras

Figure 4: Improved sequencing performance - reduction in Strand Split Artefact Reads (SSARs)

Improved sequencing performance - reduction in Strand Split Artefact Reads (SSARs). PCR-free whole genome libraries were prepared using 100 ng of Covaris-sheared human genomic DNA (NA12878) with the KAPA EvoPrep Kit, Supplier I, Supplier Q and Supplier W, following each supplier’s instructions for use. The ultra low percentage of SSARs present for KAPA EvoPrep Kit was equivalent to Supplier I and Supplier Q, with Supplier W having the highest percentage of SSARs present. SSARs represent chimeric reads that appear to be derived from non-contiguous portions of the genome.*

*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/nat/gky1142.

Improved sequencing performance - reduction in Strand Split Artefact Reads (SSARs)

Figure 5: Improved sequencing performance - KAPA EvoPrep workflow had no impact on overall coverage depth and uniformity, or GC bias - normalized coverage across the GC spectrum

PCR-free whole genome libraries were prepared using 100 ng of Covaris-sheared human genomic DNA (NA12878) with the KAPA EvoPrep Kit, Supplier I, Supplier Q and Supplier W, following each supplier's instructions for use. The KAPA EvoPrep Kit had uniform coverage and low AT- or GC-bias. Supplier I had slightly better uniform coverage in the GC-rich region and Supplier Q had lumpy coverage and coverage hotspots i.e., over-representation of AT-rich region. This could require more sequencing to be performed to achieve the required coverage for these regions, which increases cost and turnaround times.

Improved sequencing performance - KAPA EvoPrep workflow had no impact on overall coverage depth and uniformity, or GC bias - normalized coverage across the GC spectrum

Figure 6: Improved sequencing performance - lower duplication count

PCR-free whole genome libraries were prepared using 100 ng of Covaris-sheared human genomic DNA (NA12878) with the KAPA EvoPrep Kit, Supplier I, Supplier Q and Supplier W, following each supplier's instructions for use. KAPA EvoPrep Kit had very low duplication count compared to Supplier Q and Supplier W, thereby having more unique library molecules present*.

Improved sequencing performance - KAPA EvoPrep workflow had no impact on overall coverage depth and uniformity, or GC bias - normalized coverage across the GC spectrum
Improved performance with challenging sample types
  • Generate more diverse libraries from challenging sample types such cfDNA
  • Higher confidence in data generated due to reduction in sequencing artefacts present

Figure 7: Improved sequencing performance - high number of unique library molecules

Whole genome libraries were prepared using 10 ng of Covaris-sheared human genomic DNA (NA12878) with the KAPA EvoPrep Kit, Supplier I, Supplier N, Supplier Q, Supplier T and Supplier W, following each supplier’s instructions for use. KAPA EvoPrep Kit had higher number of unique library molecules present compared to Supplier N, Supplier Q, Supplier T and Supplier W, resulting in higher library diversity and data confidence*.

*Source: McNulty, et al. 2020. Impact of reducing DNA input on next-generation sequencing library complexity and variant detection. The journal of Molecular Diagnostics, Volume 22, Issue 5, May 2020, Pages 720-727

Improved sequencing performance - high number of unique library molecules

Figure 8: Improved sequencing performance - reduction in Chimeras

Whole genome libraries were prepared using 10 ng of Covaris-sheared human genomic DNA (NA12878) with the KAPA EvoPrep Kit, Supplier I, Supplier N, Supplier Q, Supplier T and Supplier W, following each supplier's instruction for use. The KAPA EvoPrep Kit had a low percentage of Chimeras present, resulting in higher data confidence*, with Supplier N, Supplier Q and Supplier W having a higher percentage of Chimeras present.

*Source: Chen, et al. (2024) Characterization and mitigation of artifacts derived from NGS library preparation due to structure-specific sequences in the human genome. BMC Genomics (2024) 25:227 https://doi.org/10.1186/s12864-024-10157-w

Improved sequencing performance - reduction in Chimeras
Exceptional performance and sequencing results
  • Excellent utilization of sequencing throughput as evidenced by high uniformity
  • Greater target coverage and unique molecule recovery from cfDNA vs other suppliers

Figure 9: Improved sequencing performance - Uniformity by % Bases within 2-fold of median coverage

10 ng of cfDNA was used to prepare triplicate libraries with the KAPA EvoPrep Kit, Supplier I, Supplier N, Supplier Q, Supplier T and Supplier W, following each supplier’s instructions for use. Libraries were enriched with the KAPA HyperCap Oncology Panel (214 Kb), following the KAPA HyperCap cfDNA Evolved workflow instructions. The KAPA EvoPrep Kit had one of the highest percentage of coverage uniformity compared to other suppliers, thereby showcasing the optimal utilization of sequencing throughput by higher uniformity.

Improved sequencing performance - Uniformity by % Bases within 2-fold of median coverage

Figure 10: Improved sequencing performance - % Target bases by at least 1000X

10 ng of cfDNA was used ot prepare triplicate libraries with the KAPA EvoPrep Kit, Supplier I, Supplier N, Supplier Q, Supplier T and Supplier W, following each supplier's instructions of use. Libraries were enriched with the KAPA HyperCap Oncology Panel (214 Kb), folowing the KAPA HyperCap cfDNA Evolved workflow instructions. The KAPA EvoPrep Kit had the highest percentage of target coverage at ≥ 1000X compared to other suppliers.

Improved sequencing performance - %Target bases by at least 1000X

Figure 11: Improved sequencing performance - Genome equivalent recovery rate (%)

10 ng of cfDNA was used to prepare triplicale libraries with the KAPA EvoPrep Kit, Supplier I, Supplier N, Supplier Q, Supplier T and Supplier W, following each supplier's instructions for use. Libraries were enriched with the KAPA HyperCap Oncology Panel (214 Kb), following the KAPA HyperCap cfDNA Evolved workflow instructions, KAPA EvoPrep Kit had higher unique molecule recovery from 10 ng of cfDNA compared to Supplier N, Supplier Q, Supplier T and Supplier W, resulting in higher data confidence*.

*Source: McNulty, et al. 2020. Impact of reducing DNA input on next-generation sequencing library complexity and variant detection. The journal of Molecular Diagnostics, Volume 22, Issue 5, May 2020, Pages 720-727

Improved sequencing performance - Genome equivalent recovery rate (%)
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*Data on file. All graphic data is on file, unless otherwise noted.

Specifications

  • Compatible Platform

    All Illumina sequencing instruments

  • Library Type

    DNA

  • Starting Material

    Fragmented DNA, cfDNA, FFPET DNA, PCR amplicons

  • Input amount

    0.1 ng - 500 ng
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Cap or Plate colour Component Name

Included in KAPA EvoPrep Kits

(10154039001) (10153806001)
(10096039001) (10153814001)

Included in KAPA EvoPrep Kits (VK*)

(10212233702) (10212250702)
(10212268702) (10212276702

Included in KAPA EvoPrep Kits (PCR-free)

(10153849001) (10153857001)
(10153865001) (10154284001)
Blue End Repair and A-Tailing ReadyMix Yes Yes Yes
Yellow Ligation ReadyMix Yes Yes Yes
Green KAPA HiFi HotStart ReadyMix (2X) Yes Yes No
Green KAPA Library Amplification Primer Mix (10X) No Yes No
Kits should be stored at -15 ̊C to -20 ̊C | *Virtual kits
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Roche Cat. No Description Kit Size (reactions)
10154039001 KAPA EvoPrep Kit, 24 rxn 24
10096039001 KAPA EvoPrep Kit, 96 rxn 96
10153849001 KAPA EvoPrep Kit, 384 rxn 384
10153865001 KAPA EvoPrep Kit, 96 rxn, plate format 96
10153806001* KAPA EvoPrep Kit, 24 rxn (PCR-free) 24
10153814001* KAPA EvoPrep Kit, 96 rxn (PCR-free) 96
10153857001* KAPA EvoPrep Kit, 384 rxn (PCR-free) 384
10154284001* KAPA EvoPrep Kit, 96 rxn, plate format (PCR-free) 96
10212233702** KAPA EvoPrep Kit + KAPA Library Amplification Primer Mix (10X), 24 rxn 24
10212250702** KAPA EvoPrep Kit + KAPA Library Amplification Primer Mix (10X), 96 rxn 96
10212268702** KAPA EvoPrep Kit + KAPA Library Amplification Primer Mix (10X), 384 rxn 384
10212276702** KAPA EvoPrep Kit + KAPA Library Amplification Primer Mix (10X), 96 rxn, plate format 96
There are no Kit Codes for the items above | * KAPA Library Amplification Primer Mix (10X) not included | ** Virtual kits
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Kit Code Roche Cat. No Description Kit Size (reactions)
KK8007 08963835001 KAPA HyperPure Beads (5 mL) 5 mL
KK8008 08963843001 KAPA HyperPure Beads (30 mL) 30 mL
KK8009 08963851001 KAPA HyperPure Beads (60 mL) 60 mL
KK8011 08963878001 KAPA HyperPure Beads (4 x 60 mL) 4 x 60 mL
KK8010 08963860001 KAPA HyperPure Beads (450 mL) 450 mL
KK8727 08861919702 KAPA Unique Dual-Indexed Adapters Kit (15μM) 96 adapters x 20μL each 
KK8721 08278539001 KAPA Adapter Dilution Buffer (25 mL) 25 mL
N/A 09063781001 KAPA Universal Adapter, 15μM 960 μL 96-192* samples
N/A 09063790001 KAPA Universal Adapter, 15μM 960 μL 384-768* samples
N/A 09329862001 KAPA Universal UMI Adapter,960 μL 96-192* samples
N/A 09329889001 KAPA Universal UMI Adapter,4x960 μL 384 samples
N/A 09134336001 KAPA UDI Primer Mixes,1-96, 96 rxn 96 samples
N/A 09329838001 KAPA UDI Primer Mixes, 97-192, 96 rxn 96 samples
N/A 09329846001 KAPA UDI Primer Mixes,193-288, 96 rxn 96 samples
N/A 09329854001 KAPA UDI Primer Mixes, 289-384, 96 rxn 96 samples
*Workflow dependent

Additional pack sizes are available. Please speak to your local sales representative about how we can make it possible for you.

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FAQ

FAQ

  • Whole-genome shotgun sequencing
  • Whole-genome or targeted sequencing, using KAPA HyperCap Workflows or KAPA HyperPETE Workflows
  • End repair and A-tailing, which produces end-repaired, 5′-phosphorylated, 3′-dA-tailed, dsDNA fragments
  • Adapter ligation, during which dsDNA adapters with 3′-dTMP overhangs are ligated to 3′-dA-tailed library fragments
  • Library amplification (optional), which employs high-fidelity, low-bias PCR to amplify library fragments carrying appropriate adapter sequences on both ends

The library construction process, from end repair and A-tailing to final library, can be performed in ~ 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 after 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 1 month before amplification, target capture and/or sequencing. To avoid degradation, always store DNA in a buffered solution (10 mM Tris-HCl, pH 8.0 – 8.5) when possible, and minimise the number of freeze-thaw cycles.

  • If you are performing a cleanup between shearing and end repair, shear in 10 mM Tris-HCl (pH 8 or 8.5) + 1 mM EDTA
  • If you are not performing a cleanup between shearing and end repair, shear in 10 mM Tris-HCl (pH 8 or 8.5) + 0.1 mM EDTA
  • Never shear DNA in water

KAPA Adapters are recommended for use with the KAPA EvoPrep Kits. For assistance with third-party adapter compatibility and ordering, please contact Technical Support for guidelines on the formulation of user-supplied library amplification primers.

Please refer to the KAPA UDI Adapters and KAPA HyperPlex 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 the 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 EvoPrep 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 the amount of adapter-ligated library retrieved after the post-ligation cleanup is sufficient for the next process (e.g. direct sequencing), and the library molecules contain all the adapter motifs needed for that process, library amplification may be omitted. This further streamlines the workflow and reduces the overall library preparation time to <2 hours. The high conversion efficiency achievable with the KAPA EvoPrep Kit enables PCR-free workflows with ≥ 50 ng input DNA. KAPA EvoPrep Kits without amplification reagents (10153806001, 10153814001, 10153857001 and 10154284001) are available for PCR-free workflows.

No, the KAPA Library Amp Primers will be available separately. This is different from our on-market KAPA HyperPrep and HyperPlus Kits. The rationale here is that because the KAPA EvoPrep Kits are optimized for WGS and target sequencing workflows, when combined with our KAPA HyperCap Evolved workflow v4.x, the customer will use the truncated KAPA HyperPlex adapters with indexing by PCR (i.e., KAPA Universal Adapter + KAPA UDI Primer Mixes). Also, we know that many customers use their own 3rd party adapters with indexing by PCR.

KAPA EvoPrep Kits contain a highly optimised Library Amplification Primer Mix, designed to eliminate or delay primer depletion during library amplification reactions performed with KAPA HiFi DNA polymerase. The primers in the mix are based on the P5 and P7 Illumina flow cell sequences, and are suitable for the amplification of libraries prepared with full-length adapters. User-supplied primer mixes may be used in combination with incomplete or custom adapters. Please contact us for guidelines on the formulation of user-supplied library amplification primers.

If cycled to completion (not recommended) a single 50 μL KAPA HiFi library amplification 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.

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 readymixes provided in this kit are temperature sensitive, and appropriate care should be taken during shipping and storage. KAPA EvoPrep Kits are shipped on dry ice or ice packs, depending on the country of destination. Upon receipt, immediately store the readymixes at -15°C to -25°C 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.

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