KAPA EvoPlus V2 Kits

Overview

The KAPA EvoPlus V2 Kits offer improved fragmentation performance, insensitivity to fragmentation inhibitors, better conversion efficiency, and reduced sequencing artefacts* through our streamlined and fully automatable workflow. Bringing a new and upgraded enzymatic fragmentation and library prep solution to the market will enable researchers to achieve higher confidence with increased sequencing efficiency.

The KAPA EvoPlus V2 Kits offer a complete library preparation solution when combined with KAPA Adapters and KAPA HyperPure Beads (sold separately) and a complete target enrichment solution when combined with KAPA HyperCap or KAPA HyperPETE workflows. The kits are compatible with the Illumina sequencing platform and have been qualified with automation methods.

Features and Benefits of KAPA EvoPlus V2 Kits*

• Simplified, streamlined, and automatable workflow with ReadyMix reagents, available in tube and plated format to increase efficiencies and convenience
• Compatible with a wide range of sample types, buffers and inputs, and flexible with respect to fragment size, adapter design, and library amplification
• Designed to increase library conversion rates with the KAPA EvoT4 DNA Ligase in the Ligation ReadMix, for higher sensitivity and confident variant detection particularly in challenging FFPET DNA samples
• Drastically reduced sequencing artefacts, with insensitivity to inhibitors, fully tunable fragmentation, and improved library prep performance
  

*Compared to earlier KAPA DNA Libray Prep chemistries. Data on file.

• Library insert sizes adjustable by varying fragmentation time
• Reproducible insert sizes across a range of GC content and DNA input amounts
• No impact to fragmentation - insensitive to EDTA (up to 2 mM), as well as numerous other inhibitors

See performance data showing tunability and consistency in DNA library insert sizes with samples from different input amounts and DNA dilution buffer.

Figure 2:  The KAPA EvoPlus V2 chemistry enables tunable enzymatic fragmentation

Human genomic DNA (100 ng) was fragmented at 37°C for different periods of time (5 – 30 min) to achieve mode library insert sizes ranging from approximately 250 – 1000 bp. The KAPA EvoPlus V2 workflow was completed without any size selection using full-length adapters (KAPA UDI Adapters) and 3 cycles of amplification to enable visualisation. Electropherograms were generated with LabChip GX Touch NGS 3K Assay.

Figure 3: KAPA EvoPlus V2 chemistry enables reproducible insert sizes across a range of DNA input amounts

0.1 ng - 500 ng of high-quality human genomic DNA eluted in 10 mM Tris-HCl, pH 8.0 was fragmented for 25 minutes and used to prepare libraries with KAPA Universal Adapters at the recommended adapter:insert molar ratio following the KAPA EvoPlus V2 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.

Figure 4: The KAPA EvoPlus V2 chemistry enables reproducible enzymatic fragmentation across DNA diluted in different buffer types

100 ng of high-quality human genomic DNA eluted in 10 mM Tris-HCl, pH 8.0, 10 mM Tris-HCl, pH 8.0 + 1 mM EDTA or PCR grade water was fragmented for 15 minutes and used to prepare libraries with KAPA UDI Adapters at the recommended adapter:insert molar ratio following the KAPA EvoPlus V2 Kit Instructions for Use. Libraries were amplified for 3 cycles to enable visualization. Electropherograms were generated with LabChip GX Touch NGS 3K Assay.

• KAPA Evo Plus V2 Kits deliver high-performing enzymatic fragmentation without drawbacks
• Improved sequencing metrics allow higher confidence in data due to reduction in sequencing artefacts

Figure 6: Improved sequencing performance - minimal start site bias

PCR-free whole genome libraries were prepared using 100 ng of human genomic DNA (NA12878) with the KAPA EvoPlus V2 Kit, Supplier I, Supplier IL, Supplier Q, Supplier T and Supplier W, following each supplier’s instructions for use. The KAPA EvoPrep Kit was included to present gold-standard* (mechanical fragmentation). The KAPA EvoPlus V2 Kit had the least start site bias compared to other Suppliers, resulting in higher data confidence**, with the most start site bias associated with Supplier IL.

*Deurenberg et al. (2017) Application of next generation sequencing in clinical microbiology and infection prevention. Journal of Biotechnology 243, 16-24.

** 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, Pages 720-727

Figure 7: Improved sequencing performance - better soft-clip distribution

PCR-free whole genome libraries were prepared using 100 ng of human genomic DNA (NA12878) with the KAPA EvoPlus V2 Kit, Supplier I, Supplier IL, Supplier Q, Supplier T and Supplier W, following each supplier’s instructions for use.  The KAPA EvoPlus V2 Kit had the lowest count of softclipped reads compared to other Suppliers, with Supplier IL  having the highest count of softclipped reads.

Figure 8: Improved sequencing performance - reduction in Strand Split Artefact Reads (SSARs)

PCR-free whole genome libraries were prepared using 100 ng of human genomic DNA (NA12878) with the KAPA EvoPlus V2 Kit, Supplier I, Supplier IL, Supplier Q, Supplier T and Supplier W, following each supplier’s instructions for use.  The KAPA EvoPlus V2 Kit had the lowest percentage of SSARs present compared to other Suppliers, with Supplier Q  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/nar/gky1142.

Figure 9: Improved sequencing performance - reduction in Chimeras

PCR-free whole genome libraries were prepared using 100 ng of human genomic DNA (NA12878) with the KAPA EvoPlus V2 Kit, Supplier I, Supplier IL, Supplier Q, Supplier T and Supplier W, following each supplier’s instructions for use.  The KAPA EvoPlus V2 Kit had a lower percentage of Chimeras present, resulting in higher data confidence* compared to Supplier I, Supplier Q, Supplier T and Supplier W.

*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

• High specificity and percent of target coverage at ≥200x 
• Reduction in the presence of sequencing artefacts

Figure 11: Improved sequencing performance - % Target Bases by at least 200X

50 ng of low quality FFPET DNA was used to prepare triplicate libraries with the KAPA EvoPlus V2 Kit and mechanical fragmentation kits of 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 FFPET Evolved workflow instructions. The KAPA EvoPlus V2 Kit had very high percentage of target bases covered by at least 200X compared to other mechanical fragmentation suppliers, thereby showcasing the optimal utilization of sequencing throughput.

Figure 12: Improved sequencing performance - reduction in sequencing artefacts

50 ng of low quality FFPET DNA was used to prepare triplicate libraries with the KAPA EvoPlus V2 Kit and mechanical fragmentation kits of 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 FFPET Evolved workflow instructions. The KAPA EvoPlus V2 Kit had the lowest percentage of Chimeras present, resulting in higher data confidence* compared to mechanical fragmentation suppliers (Supplier I, Supplier N, Supplier Q, Supplier T and Supplier W).

*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