Flow-Cycle Sequencing for Accurate Short Variant Calling
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Solution Overview
Problem
Existing sequencing methods for detecting genetic variants are inefficient and prone to errors, particularly in single-signal sequencing, which can be overcome by high-depth sequencing but is costly and time-consuming.
Innovation Solution
A method for detecting short genetic variants using non-terminating nucleotides in separate nucleotide flows, generating sequencing data sets with flow signals at specific positions, and determining match scores to accurately call the presence or absence of variants, potentially using different flow-cycle orders for enhanced accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If high-depth sequencing is used to overcome sequencing errors, then measurement precision is improved, but cost and time consumption increase
Solution Approach 1:
The patent segments the sequencing process into multiple flow cycles with different flow-cycle orders, where each cycle provides independent sequencing data. By analyzing multiple segmented data sets from different flow orders, the system achieves high measurement precision without requiring excessive sequencing depth, thus reducing time consumption while maintaining accuracy.
Solution Approach 2:
The patent introduces a new dimension of analysis by examining sequencing data across multiple flow-cycle orders rather than relying on a single sequencing run. This dimensional expansion allows the system to distinguish true variants from errors more effectively, improving measurement precision without proportionally increasing sequencing depth or time.
2Manufacturing precision
If reversible-terminator sequencing-by-synthesis is used, then manufacturing precision is improved, but device complexity increases
Solution Approach 1:
Instead of using reversible terminators that require complex chemistry and multiple washing steps, the patent inverts the approach by using non-terminating nucleotides with flow-based selective incorporation. This inversion simplifies the sequencing chemistry while maintaining precision through the flow-cycle order control mechanism, reducing device complexity.
Solution Approach 2:
The patent changes the key parameter from chemical termination mechanisms to flow-order control parameters. By controlling which nucleotide flows are provided in which order rather than using chemical terminators, the system achieves base calling accuracy without the complex chemistry required by reversible-terminator methods, thus reducing device complexity.
3Ease of operation
If single-signal sequencing is used, then ease of operation is improved, but reliability deteriorates
Solution Approach 1:
The patent implements a feedback mechanism by comparing sequencing results across multiple flow-cycle orders. The system uses this feedback to distinguish true variants from sequencing errors, improving reliability while maintaining ease of operation. The match score calculation provides quantitative feedback that guides variant calling decisions.
Solution Approach 2:
The patent creates multiple copies of sequencing data by running the same template through different flow-cycle orders. These copied data sets are then compared to identify consistent signals that represent true variants, improving reliability without complicating the operation of the sequencing system itself.
Data Source
AI summary
Methods for detecting a short genetic variant in a test sample are described herein. In some exemplary methods, the short genetic variant is called using one or match scores, which are determined using one or more sequencing data sets obtained from a test nucleic acid molecule, wherein the test sequencing data sets are determined by sequencing the test nucleic acid molecule using non-terminating nucleotides provided in separate nucleotide flows according to a flow-cycle order. Also described herein are methods of sequencing a test nucleic acid molecule using two or more different flow-cycle orders and/or extended flow cycle orders having five or more nucleotide flows per flow cycle.