Rolling Circle Amplification for Rare Variant Detection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for detecting rare sequence variations in nucleic acid sequencing face challenges due to high error frequencies and difficulties in distinguishing rare variants from sequencing errors, especially in complex populations and low-frequency variants.
Innovation Solution
The method involves generating amplicons with multiple copies of a target polynucleotide using rolling circle amplification, enriched through specific primer extension and amplification conditions, allowing for sensitive detection of rare sequence variants and gene fusion events by sequencing these amplicons.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If rolling circle amplification is used to generate concatemers, then amplification of target sequences is achieved, but short concatemers are amplified disproportionally compared to longer concatemers
Solution Approach 1:
The patent modifies the amplification parameters by using a two-step PCR approach with different annealing temperatures. The first PCR uses a lower annealing temperature (50-65°C) to amplify all concatemer lengths, while the second PCR uses a higher annealing temperature (60-75°C) to selectively amplify longer concatemers, thereby changing the parameter distribution to favor uniform length representation
Solution Approach 2:
The patent performs a preliminary PCR amplification step before the enrichment step. This first PCR generates sufficient amplification products of various concatemer lengths, which then serve as templates for the second enrichment PCR that selectively amplifies longer concatemers, ensuring adequate material is available before applying the length-selective conditions
2Productivity
If standard high throughput sequencing is used, then large scale parallel sequencing is achieved, but error rates of 0.1-1% make detection of rare sequence variants difficult
Solution Approach 1:
The patent generates multiple copies of the target sequence by rolling circle amplification to form concatemers containing multiple repeats of the target sequence. By sequencing these concatemers and requiring consensus across multiple copies, the method distinguishes true rare variants from single-base sequencing errors, thereby improving measurement precision while maintaining high throughput
Solution Approach 2:
The patent segments the target sequence into multiple copies within concatemers. By analyzing multiple segmented copies of the same target sequence and requiring consensus, the method can identify rare variants that appear consistently across copies while filtering out random sequencing errors that affect only single copies
3Productivity
If conventional RCA methods are used, then amplification of target sequences is achieved, but subsequent analysis of longer concatemers becomes more difficult
Solution Approach 1:
The patent changes the amplification parameters by using a two-step PCR approach with different annealing temperatures. The first PCR uses lower stringency to amplify all products, while the second enrichment PCR uses higher stringency (60-75°C annealing) to selectively amplify longer concatemers, making them the dominant population for subsequent analysis and reducing complexity
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 significantly reduces sequencing errors and enhances the detection of rare sequence variants, improving the identification of low-frequency mutations and gene fusions in complex samples.
Implementation Method 1
a polymerase that extends the first primer and the second primer to produce amplified polynucleotides
Implementation Method 2
the first primer specifically hybridizes to sequence A of the target sequence via sequence complementarity between sequence A and sequence A'
Data Source
AI summary
In some aspects, the present disclosure provides methods for enriching amplicons, or amplification products, comprising a concatemer of at least two or more copies of a target polynucleotide. In some embodiments, a method comprises sequencing the amplicons comprising at least two or more copies of a target polynucleotide. In some embodiments, the target polynucleotides comprise sequences resulting from chromosome rearrangement, including but not limited to point mutations, single nucleotide polymorphisms, insertions, deletions, and translocations including fusion genes. In some aspects, the present disclosure provides compositions and reaction mixtures useful in the described methods.