Variant-Selective Nucleic Acid Probes for Rare Mutation Sequencing
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Solution Overview
Problem
Current methods for targeted detection of low-frequency variants in nucleic acid sequencing are inefficient, leading to high costs and difficulty in detecting rare variants due to enrichment of wild-type molecules, which reduces sensitivity and specificity.
Innovation Solution
The use of probe nucleic acid molecules with differential complementarity to selectively isolate and detect target nucleic acids by employing techniques such as hybridization-capture and next-generation sequencing, utilizing enzymes and chemical reactions to differentially modify probes based on complementarity, allowing for error-detection and error-correction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If hybridisation-capture is used to enrich target regions, then target region coverage is improved, but variant molecule enrichment is not achieved resulting in majority wild-type reads
Solution Approach 1:
The patent applies local quality by designing probes with specific differential complementarity at local positions (mismatch positions) to distinguish variant molecules from wild-type molecules. The probes have perfect complementarity to variant sequences at critical positions while having mismatches to wild-type sequences, enabling selective enrichment of variant molecules within the captured target regions.
Solution Approach 2:
The patent changes the complementarity parameter of probes at specific positions to achieve differential binding. By introducing mismatches at predetermined positions in the probes, the binding affinity is selectively reduced for wild-type molecules while maintaining high affinity for variant molecules, thereby enabling variant-specific enrichment during hybridisation-capture.
2Adaptability or versatility
If standard NGS library preparation is used, then library complexity is maintained, but sensitivity for low-frequency variants is reduced due to wild-type enrichment
Solution Approach 1:
The patent performs preliminary action by pre-designing probes with differential complementarity before the hybridisation-capture step. These probes are prepared in advance with specific mismatch patterns that will selectively bind to variant molecules, enabling pre-enrichment of variants before sequencing library preparation and thereby improving sensitivity for low-frequency variants.
Solution Approach 2:
The patent segments the probe design into multiple regions including target-specific regions and differential complementarity regions with mismatches. This segmentation allows the probe to simultaneously maintain binding to target regions while introducing selectivity through localized mismatch patterns, enabling both library complexity maintenance and variant-specific enrichment.
3Measurement precision
If duplex sequencing is used to increase specificity, then sequencing accuracy is improved, but sensitivity is reduced due to molecular bottlenecks
Solution Approach 1:
The patent performs preliminary enrichment of variant molecules through differential probe hybridisation before sequencing. By pre-concentrating variant molecules using probes with differential complementarity, the method reduces the need for extensive molecular amplification and bottleneck steps, thereby maintaining both high specificity and sensitivity without requiring duplex sequencing.
Solution Approach 2:
The patent extracts variant molecules from the mixture by selective hybridisation to differential probes. This extraction step isolates variant molecules from wild-type background before sequencing, achieving high specificity without requiring the complex duplex sequencing protocol and its associated molecular bottlenecks, thereby preserving sensitivity.
4Measurement precision
If more sequencing reads are performed to detect rare variants, then detection sensitivity is improved, but cost and time increase
Solution Approach 1:
The patent performs preliminary enrichment of variant molecules using differential probe hybridisation before sequencing. This pre-enrichment step concentrates rare variants in the library, reducing the total number of sequencing reads required to achieve sufficient coverage and statistical power for detecting low-frequency variants, thereby improving cost and time efficiency.
Solution Approach 2:
The patent changes the enrichment parameter by using differential probe hybridisation to selectively amplify variant molecule representation in the sequencing library. This parameter change increases the allele frequency of variants in the sequenced population, reducing the sequencing depth required to detect rare variants and thereby improving productivity.
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
This approach reduces the number of sequencing reads required, enhances sensitivity for low-frequency variant detection, and enables accurate sequencing data by preferentially selecting probes that differentiate between desired and non-desired nucleic acids.
Implementation Method 1
employing differential complementarity of probe nucleic acid molecules to target nucleic acids molecules to selectively isolate and detect targets
Data Source
AI summary
Provided herein are compositions and methods for the analysis of nucleic acids. In particular, provided herein are compositions and methods employing differential complementarity of probe nucleic acid molecules to target nucleic acids molecules to selectively isolate and detect targets or probes as an indication of the presence of and/or amount of a particular target nucleic acid molecule in a sample.