NGS Library Normalization for Fast Equimolar Pooling
Find Innovative SolutionsGenerate Solutions
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 unbalanced sequencing data when libraries are pooled.
Innovation Solution
A novel PCR-based and enzyme-based normalization method that produces equimolar NGS library concentrations independently of insert size, using limited primer or probe concentrations to achieve precise molar normalization in a single or few 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 incorporates quantification primers during the library preparation PCR amplification step itself, rather than performing a separate qPCR assay. The primers are designed to be incorporated into the library molecules during the initial amplification, enabling subsequent rapid quantification by simple fluorescence measurement without requiring serial dilution, standard curves, or multiple pipetting steps
Solution Approach 2:
The patent combines the library amplification and quantification primer incorporation into a single PCR reaction. The quantification primers are merged with the library preparation process, eliminating the need for a separate quantification protocol and reducing both time and operational complexity while maintaining accurate quantification capability
2Ease of operation
If chip electrophoresis or fluorometric methods are used for library quantification, then the procedure is simplified, but these methods cannot distinguish functional from non-functional molecules in PCR-free libraries
Solution Approach 1:
The patent introduces quantification primers as an intermediary element that binds specifically to functional library molecules during the PCR amplification step. These primers serve as a mediator that enables subsequent fluorescence-based detection to distinguish functional molecules from non-functional ones, combining the simplicity of fluorometric methods with the accuracy of functional molecule quantification
Solution Approach 2:
The patent applies local quality by making the quantification capability specific to functional molecules through the use of primers that only bind to molecules with both adapters properly ligated. The quantification property is localized to functional molecules rather than measuring total DNA, enabling accurate distinction between functional and non-functional library molecules while maintaining procedural simplicity
3Manufacturing precision
If manual concentration adjustment is performed for each library when pooling samples, then sequencing balance can be controlled, but the process is time-consuming and requires hands-on adjustment
Solution Approach 1:
The patent enables libraries to self-normalize to equimolar concentrations through the PCR amplification process itself. By incorporating quantification primers during amplification and using the resulting fluorescence signals, the system automatically determines the appropriate pooling ratios without requiring manual concentration measurement and adjustment for each individual library
Solution Approach 2:
The patent implements feedback by using the fluorescence signal from quantification primers bound to amplified library molecules to directly inform the pooling strategy. The quantification data feeds back into the library pooling decision-making process, enabling automated determination of equimolar pool composition without manual intervention while maintaining precise control over sequencing balance
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 enable fast, accurate, and automated normalization of NGS libraries, ensuring consistent sequencing output and reducing data imbalance by eliminating excess library quantities, compatible with various sequencing platforms.
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
The method requiring several steps is an enzyme-based normalization which entails: a. PCR amplification to an excess quantity of each NGS library using pre-Normalization primers (pre-N primers)
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.