NORMALASE™
4Pages

SWIFT NORMALASE™ KITS Revolutionary NGS Library Normalization Technology Introduction The Swift Normalase Kits offer a novel enzymatic library normalization technology that consolidates DNA or RNA library normalization and pooling for loading on Illumina® sequencing platforms. The Normalase workflow eliminates the need for library concentration adjustment prior to library pooling, resulting in optimal cluster density and library balance (Figure 1). The Swift normalization method can easily be integrated into standard library preparation protocols to improve turnaround time and loading accuracy for NGS laboratories. The library selection and enzymatic normalization steps of the Normalase workflow are designed for workflows that consistently produce amplified library yields 3x the target normalization amount following library amplification with Normalase primers (e.g., 12 nM yield for 4 nM normalization). This workflow does not introduce a second PCR; instead, it replaces the primers in conventional library amplification, either terminal or indexing. Swift Normalase Kits offer a fast, scalable library normalization workflow for high-throughput laboratories. Normalase Workflow Highlights: Saves time and increases throughput Uniform sample processing to generate a balanced, multiplexed library pool Reduces variability to save on sequencing costs Better balanced pools allow higher multiplexing per run and fewer re-sequencing costs Flexible design for many workflows Compatible with diverse library preparation methods to produce evenly balanced sequence data Indexed Adapters Figure 1 (right): The Swift Normalase workflow begins after NGS library adapter ligation, using either full length indexed adapters or truncated adapters, where Normalase PCR primers are used to amplify the libraries to above the minimum threshold and condition the libraries for downstream Normalase enzymo/ogy. For full length indexed adapter libraries, Swift Normalase terminal primers are used, for truncated adapter libraries, Swift Combinatorial Dual indexing Normalase primers are used. Amplified and conditioned NGS libraries are then individually incubated for 15 minutes with the Normalase i master mix to enzymatically select a specified molarity of each NGS library. After Normalase i, each library is pooled using equal volumes into a single tube and incubated for 15 minutes with the Normalase H mastermix which enzymatically normalizes each NGS library to the specified selected molarity. The result of Normalase is a balanced multiplexed NGS library pool ready for sequencing. Normalized Library Pool (4nM) Normalized Library Pool (4nM)

Swift Normalase Kits (continued) Highly Reproducible DNA Library Normalization and Robust Performance Across Variable Insert Sizes Cluster Density (K/ mm 2) Library Balance Table 1 (Above): Expected and consistent cluster density generation using Mi Seq ® v2 chemistry at 12 pM from library pools normalized to 4 nM using Normalase. Better Normalization Compared to Conventional Methods Figure 2 (Above): Thirty-two Swift 2S Turbo libraries were generated with fulllength indexed adapters between two users (n=16/user) with 1 to 250ng inputs of NA12878 gDNA. Post-Normalase PCR libraries were...

Swift Normalase Kits (continued) Normalase Qubit Figure 5 (Above): Swift 2S Turbo libraries were made from 5ng of MSA-1000, an equal mass mix of 10 different bacterial strain genomes with varying GC%. Two libraries were either amplified and indexed with Swift CD Indexing Normalase primers (plum) or Swift CD Indexing primers (blue). Normalase conditioned libraries were normalized to 4nM and pooled using Normalase, while standard libraries were Qubit quantified and pooled at 4nM. Libraries were co-sequenced on the Illumina MiniSeq using High Output reagents and 150 PE sequencing. Across...

Table 3 (Above): Coverage metrics of the top 1000 expressed transcripts demonstrating no differences between qPCR manual pooling and Normalase. Figure 6 (Right): Venn diagram of the Top 1000 transcripts detected in each sample demonstrating no significant differences in transcripts detected Post-Library amplification molarity required prior to Normalase 3x target molarity in 20 qL Normalized library concentration post Normalase 4 nM Library balance within a pool Coefficient of variation < 10% Library compatibility Swift Indexing kit compatibility System compatibility • Libraries with...