Rolling Circle Amplification for Rare Sequence Variant Detection
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Solution Overview
Problem
Current large-scale parallel nucleic acid sequencing techniques face challenges in detecting rare sequence variants due to high error frequencies, leading to false positives and difficulties in identifying low-frequency genetic variations, which are crucial for applications such as contaminant detection in space exploration, food monitoring, and early disease diagnosis.
Innovation Solution
The method involves rolling circle amplification of circularized polynucleotides, where individual polynucleotides are circularized using a ligase enzyme, degraded, and then amplified without purification, followed by sequencing to identify sequence differences that occur in multiple circular polynucleotides or sheared fragments, ensuring accuracy in detecting rare variants.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If standard high throughput sequencing is used, then sequencing throughput is high, but error rate increases to 0.1-1% making rare variant detection unreliable
Solution Approach 1:
The method segments the sequencing process into multiple independent circularization events, where each polynucleotide is circularized separately before amplification. This segmentation allows rare variants to be detected against a background of individually processed molecules, reducing the impact of systematic errors and enabling detection at frequencies below 0.1%.
Solution Approach 2:
The patent applies preliminary circularization of polynucleotides before amplification and sequencing. By circularizing individual polynucleotides first, the method creates unique molecular identifiers that allow post-sequencing differentiation of true rare variants from sequencing errors, effectively preparing the sample in advance for high-precision variant detection.
2Device complexity
If circularization and amplification steps are performed without purification, then process complexity is reduced, but contamination from linear polynucleotides may increase
Solution Approach 1:
The patent introduces an intermediary enzymatic digestion step using exonuclease to selectively remove linear polynucleotides while leaving circularized polynucleotides intact. This intermediary step acts as a selective filter that maintains process simplicity while ensuring reliability by eliminating potential contaminants before amplification.
Solution Approach 2:
The method exploits parameter changes in polynucleotide structure (circular vs. linear) to differentiate and selectively process them. By changing the physical state from linear to circular through ligation, the patent creates a detectable and selectable difference that enables contamination control without complex purification procedures.
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 enhances the sensitivity and specificity of rare sequence variant detection, reducing error rates and improving the identification of low-frequency genetic changes, thereby supporting more reliable contaminant detection and early disease diagnosis.
Implementation Method 1
circularizing individual polynucleotides in a plurality of polynucleotides to form a plurality of circular polynucleotides using a ligase enzyme
Implementation Method 2
amplifying the circular polynucleotides after degrading the ligase enzyme to produce amplified polynucleotides
Data Source
AI summary
In some aspects, the present disclosure provides methods for identifying sequence variants in a nucleic acid sample. In some embodiments, a method comprises identifying sequence differences between sequencing reads and a reference sequence, and calling a sequence difference that occurs in at least two different circular polynucleotides, such as two circular polynucleotides having different junctions, or two different sheared polynucleotides as the sequence variant. In some aspects, the present disclosure provides compositions and systems useful in the described method.