KAPA EvoPlus V2 Kits

Figure 1: Comparison of the core KAPA EvoPlus V2 workflow to another enzymatic fragmentation library preparation kit. Both workflows are carried out in a single tube up to the library amplification step, which is optional when full-length indexed adapters are used. KAPA EvoPlus V2 ReadyMix reagents are ready-to-use; no master mix preparation is required. With KAPA EvoPlus V2, only two reagents (ReadyMix #1 & ReadyMix #2) are added to input DNA vs. the Supplier A workflow which requires additional reagents & mixing steps, thus increasing the quantity of consumables used & the number of pipetting steps. The core reaction (all non-optional steps) for KAPA EvoPlus V2 can be completed in only four pipetting steps when using full-length adapters, vs. Supplier A which requires nine. NOTES: KAPA full-length UDI adapters are only available in 96-well plates. KAPA Universal Adapters, which are used in combination with KAPA UDI Primer Mixes and require amplification, are available in plates or tubes. KAPA HyperPure Beads, UDI Adapter Kits & KAPA Library Amplification Primer Mix (10X) are sold separately.

Figure 3: KAPA EvoPlus V2 Kits enable tunable enzymatic fragmentation & reproducible input sizes, even in the presence of buffers containing inhibitors. (A) Mode library insert sizes ranging from approximately 250–1000 bp were achieved by fragmenting 100 ng high-quality human genomic DNA (hgDNA) at 37°C for 5 to 30 min. The workflow was completed without size selection using full-length KAPA UDI Adapters and KAPA UDI Primer Mixes. (B) A range of hgDNA input amounts (0.1 ng-500 ng, in 10 mM Tris-HCl, pH 8.0) was fragmented for 25 minutes & used to prepare libraries with KAPA Universal Adapters. (C) 100 ng of hgDNA in different buffer types (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 & used to prepare libraries with full-length KAPA UDI Adapters. For all libraries, the adapter:insert molar ratios were determined using the KAPA EvoPlus V2 Kit Instructions for Use & libraries were amplified ≥3 cycles to enable visualization. Electropherograms were generated with LabChip GX Touch NGS 3K Assay.

Figure 5: KAPA EvoPlus V2 Kit yields improved sequencing performance in the KAPA HyperCap FFPET target capture workflow, compared to other suppliers’ mechanical fragmentation kits. (A) The KAPA EvoPlus V2 Kit yielded the highest percentage of reads on-target compared to the other library prep kits tested. (B) The KAPA EvoPlus V2 Kit yielded a very high percentage of target bases covered by at least 200X. (C) The KAPA EvoPlus V2 Kit yielded more unique reads (as determined by UMI sequences), indicating greater library complexity and a greater recovery of unique input molecules. (D) KAPA EvoPlus V2 Kit yielded the very low chimeric read artifacts, even when compared against mechanical fragmentation library preps. Methods: For all libraries, 50 ng of low-quality FFPET DNA was used to prepare triplicate libraries following the suppliers’ instructions for use. All libraries were enriched with the KAPA HyperCap Oncology Panel (214 Kb) following the KAPA HyperCap FFPET Evolved workflow instructions. All libraries were sequenced on an Illumina NovaSeq 6000 (2x150bp) and subsampled to 150 M reads.