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Revolutionary Library Normalization Technology for NGS Laboratories
• Replaces conventional library quantification and normalization
• Reduces read-depth variation to ≤10% within a pool for more balanced libraries
• Mitigates index hopping by eliminating primer/adapter carryover
• Saves sequencing and labor costs
The Swift Normalase Kit offers a novel enzymatic library normalization technology that is augmented by the power to consolidate library normalization and pooling for loading on Illumina® sequencing platforms. The Normalase workflow eliminates the need for library quantification and concentration adjustment prior to library pooling, resulting in optimal cluster density and library balance. The Swift normalization method can easily be integrated into standard protocols to improve turnaround time loading and accuracy for NGS laboratories.
Conventional Workflow compared to Normalase Workflow
Figure 1: The first step of Normalase replaces conventional library amplification, but integrates special primers to pre-condition the libraries, which is followed by two 15 minute incubations to normalize libraries to 4 nM within a single pool.
Saves time and increases throughput
Uniform sample processing with fewer handling steps to generate balanced library representation in multiplexed sequencing.
Reduces variability to save on sequencing costs
Improved library balancing allows higher multiplexing per run, predictable read depth, reducing the need to resequence.
Flexible design for many workflows
Compatible with diverse library preparation methods and sample types to produce more evenly balanced sequence data.
Figure 2: 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 enzymology. 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 II 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.
Better Normalization Compared to Conventional Methods
Figure 3: 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 quantified with qPCR assuring the libraries met the minimum threshold. Libraries were either normalized and pooled and sequenced based on the qPCR quant or, using the same libraries, pooled and normalized using Normalase and sequenced to determine percent Reads Identified of each index (MiSeq V2 Nano 50 cycle). The coefficient of variation (CV) for the qPCR pool was 22.5% across the two users, while the CV for the Normalase pool was 9.4%. Lines are median and 95% confidence interval.
Figure 4: Ninety-six Swift 2S Turbo libraries were generated with 10ng NA12878 gDNA and amplified with Swift Normalase Combinatorial Dual Indexing primers. Libraries were pooled, Pre-Normalase, using equal volumes and the pool quantified by Qubit for loading on the Illumina MiSeq V2 50 cycle Nano flow cell to obtain percent Reads Identified from each index. The preNormalase pool CV was 15.5%, demonstrating robust and reproducible amplification using the Normalase Indexing primers. The same libraries were subjected to Normalase and normalized to 4nM, Post-Normalase, and loaded on the Illumina MiSeq V2 50 cycle Nano flow cell. The Normalase pool CV was reduced to 7.7% demonstrating robust normalization of multiplexed library pools using Swift Normalase. Lines are median and 95% confidence interval.
Normalase Conditioning Maintains Library Complexity
Figure 5: Swift 2S Turbo libraries made from 100ng of DNA consisting of a mix of three reference bacterial strains (E.coli, S.aureus, Steptomyces) mixed at unequal proportions. DNA fragmented to 350 bp were ligated with full-length indexed Y-adapters, and were either not amplified or amplified using 3, 5, and 7 cycles of PCR using the Swift HiFi Polymerase and Normalase terminal primers. All libraries were quantified and pooled based on their qPCR quantification. Libraries were cosequenced on the Illumina MiSeq using 150PE sequencing. Across bacterial genomes with varying GC% content, there was no observed difference in A) the number of read duplicates or B) genome coverage.
Libraries Normalized Using Normalase Maintain High Quality Sequencing Coverage
Figure 6: 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 bacterial genomes with varying GC% libraries, Normalase treated ibraries maintained A) insert size and B) high quality genomic coverage
Normalase Kit Compatibility
*Swift Accel-Amplicon and Swift Amplicon Kits are not compatible with this Normalase workflow, however, an alternate workflow is available. Please contact us for more information.