NGS Library Normalization Using Limiting Primers for Equimolar Pooling
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Solution Overview
Problem
Current methods for quantifying Next-Generation Sequencing (NGS) library concentration are inaccurate and time-consuming, particularly for PCR-free libraries, and require manual adjustment, leading to inconsistent and suboptimal sequencing results due to variations in library molarity and insert size distribution.
Innovation Solution
A novel PCR-based and enzyme-based normalization method that achieves equimolar NGS library concentration independently of insert size, using limited primer or probe concentrations to normalize library quantity in a single or multiple steps, reducing hands-on time and eliminating the need for serial dilution and manual adjustments.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If qPCR is used for library quantification, then accurate quantification of functional library molecules is achieved, but the protocol takes almost 2 hours with at least 45 minutes of hands-on time
Solution Approach 1:
The patent extracts the quantification function from the complex qPCR protocol and implements it through a simplified normalization step using magnetic beads and limiting primers/probes. This separates the essential normalization need from the time-consuming qPCR measurement steps, achieving accurate quantification equivalent results in a fraction of the time.
Solution Approach 2:
The patent performs normalization as a preliminary action during library preparation rather than as a separate post-quantification step. By incorporating the normalization function into the library prep workflow using magnetic bead-based methods, the patent eliminates the need for separate qPCR quantification and manual concentration adjustment, saving significant time while maintaining accuracy.
2Ease of operation
If chip electrophoresis or fluorometric methods are used for library quantification, then the procedure is simpler and faster, but these methods cannot distinguish functional from non-functional molecules in PCR-free libraries
Solution Approach 1:
The patent introduces magnetic beads coated with capture sequences as an intermediary that specifically binds to functional library molecules containing NGS adapters. This intermediary enables the simple magnetic bead-based normalization procedure to selectively capture and quantify only functional molecules, resolving the contradiction between simplicity and accuracy for PCR-free libraries.
3Productivity
If manual concentration adjustment is performed for each library when pooling samples, then sequencing output can be optimized, but the process is time-consuming and prone to errors
Solution Approach 1:
The patent implements self-service normalization where magnetic beads with limiting primers or probes automatically normalize library concentrations during the pooling process. The system performs the normalization function autonomously through the chemical reactions and magnetic bead capture, eliminating the need for manual concentration measurement and adjustment while maintaining optimal sequencing output.
4Quantity of substance
If libraries with broad size distribution are quantified by mass, then molar quantification can be determined, but the results are inaccurate due to dependence on insert size
Solution Approach 1:
The patent changes the quantification parameter from mass-based to molarity-based by using limiting primers or probes in a magnetic bead normalization step. This parameter change allows direct determination of molar concentration independent of insert size, as the normalization reaction stoichiometry directly reflects molar ratios rather than mass ratios, resolving the accuracy issue for libraries with broad size distributions.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The methods provide fast, accurate, and reproducible normalization of NGS libraries, ensuring consistent sequencing output and reducing preparation time by up to 3 hours compared to qPCR quantification, applicable to both PCR-amplified and PCR-free libraries.
Implementation Method 1
amplification of each NGS library is performed using a limited concentration of normalization primers (N-PCR primers) with accelerated annealing kinetics and amplification conditions for complete primer utilization during the PCR reaction
Implementation Method 2
primers (N-PCR primers) with accelerated annealing kinetics
Implementation Method 3
incubation of each amplified library with a limiting specified molar quantity of normalization probe (N-probe) and in some embodiments a DNA ligase, where probe annealing and ligation to the 5′ or 3′ overhang selects a library fraction that is equimolar to the N-probe
Implementation Method 4
incubation of each amplified library with a limiting specified molar quantity of normalization probe (N-probe) and in some embodiments a DNA ligase, where probe annealing and ligation to the 5′ or 3′ overhang selects a library fraction that is equimolar to the N-probe
Implementation Method 5
isolation of the probe selected fraction is performed by enzymatic digestion of the non selected library fraction or by enzyme-mediated release of the probe selected fraction from solid phase immobilization
Data Source
AI summary
A bottleneck in the Next Generation Sequencing (NGS) workflow is the quantification of libraries for accurate pooling and loading of the sequencing instrument flow cell or chip. Disclosed herein are methods that improve performance and reduce time compared to existing methods.