Multiplex PCR Primer Design for Even NGS Read Distribution
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Solution Overview
Problem
Existing Next Generation Sequencing (NGS) methods face challenges in multiplex genotyping due to primer:primer interactions leading to primer dimer extension products and uneven amplification of genotyping targets, reducing throughput and efficiency.
Innovation Solution
Improved primer design methods using anti-sense oligos and computer algorithms to minimize primer:primer interactions, along with anti-sense oligos to inhibit high-performing amplicons, allowing poorer performing amplicons to produce more data, and personalized software for rapid data analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiplex amplification is performed with hundreds to thousands of target variable sequences, then genotyping throughput is improved, but primer:primer interactions occur leading to primer dimer extension products and uneven amplification
Solution Approach 1:
The patent applies preliminary anti-action by introducing anti-sense oligos before the multiplex amplification reaction. These anti-sense oligos are designed to specifically bind to and inhibit high-performing amplicons in advance, preventing them from dominating the amplification reaction. This pre-inhibition strategy allows poorer performing amplicons to produce more data, thereby achieving even reads across all targets while maintaining high throughput genotyping capability
Solution Approach 2:
The patent uses anti-sense oligos as intermediary molecules that mediate between the primers and the target sequences. These intermediaries selectively bind to high-performing amplicons, acting as a buffer that prevents primer:primer interactions and primer dimer formation. The anti-sense oligos transfer the inhibition function from direct primer:primer interactions to a controlled intermediary binding mechanism, enabling uniform amplification across hundreds to thousands of targets
2Reliability
If anti-sense oligos are used to inhibit high-performing amplicons, then read evenness is improved, but reaction complexity increases
Solution Approach 1:
The patent applies local quality by designing anti-sense oligos with specific local characteristics tailored to each high-performing amplicon. Each anti-sense oligo has a unique sequence complementary to its target amplicon, allowing selective inhibition only where needed. This localized approach ensures that only specific high-performing amplicons are inhibited while leaving other targets unaffected, achieving read evenness without unnecessarily complicating the entire reaction system
Solution Approach 2:
The patent uses parameter changes by adjusting the concentration and sequence composition of anti-sense oligos to optimize their inhibitory effect. By carefully controlling the molar ratio of anti-sense oligos to primers and adjusting their melting temperatures, the system achieves precise control over which amplicons are inhibited. This parameter optimization allows the system to manage reaction complexity while maintaining effective read evenness across multiple targets
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
Enhances throughput in NGS-based multiplex genotyping by achieving even reads for 2-10,000 amplicons and 1-100,000 DNA samples, enabling single base multiplexed sequencing and rapid analysis of NGS data, particularly in identifying low abundance rare variants in tumor cells.
Implementation Method 1
anti-sense oligos in various concentrations to inhibit high-performing amplicons from forming
Implementation Method 2
iterative calculation of predicted ΔG (or delta G, Gibbs free energy) for interactions between primers to generate a fitness score
Data Source
AI summary
The technology described herein is directed to methods of designing primers for multiplex PCR amplification. Also described herein are methods for equalization of reads in these approaches. A variation is described herein that permits single base multiplexed sequencing on an NGS platform. Also described herein are methods to rapidly analyze NGS sequencing data to automatically provide genotype or sequencing results and methods to identify and quantify low abundance rare variants in clinically relevant genes in a minority of tumor cells from a complex mixture of cells.