Duplex Sequencing for Error-Corrected Nucleic Acid Mixture Resolution
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Solution Overview
Problem
Existing methods for resolving mixed cell populations and nucleic acid mixtures are complex, expensive, and often require intact cells or high sequencing depth, especially when dealing with complex mixtures, and may not be effective with conventional next-generation sequencing (NGS) methods.
Innovation Solution
The use of Duplex Sequencing methods to generate error-corrected sequence reads by ligating adapter molecules to double-stranded DNA, sequencing both strands, and comparing them to identify correspondences, allowing for the deconvolution of nucleic acid mixtures into individual genotypes and donor sources.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If single-cell analysis approach is used to resolve mixed cell populations, then genotype distinction between individuals is improved, but device complexity and cost increase
Solution Approach 1:
The method segments the sequencing process into two independent strands (first and second strands) of the same DNA molecule. Each strand is sequenced separately and independently error-corrected, then the results are combined to provide a comprehensive genotype profile. This segmentation allows parallel processing and reduces the complexity of single-cell analysis by distributing the sequencing workload across two simpler, independent sequencing reactions rather than requiring complex single-molecule sequencing systems.
2Ease of operation
If bulk extraction and genotyping of nucleic acids is used, then procedure simplicity is improved, but sequencing depth and accuracy requirements increase
Solution Approach 1:
The method implements feedback through error correction by sequencing both the first and second strands of the same DNA molecule and using the results to correct errors in either strand. The sequencing results from one strand serve as feedback to verify and correct potential errors in the other strand, thereby maintaining high sequencing accuracy while using simpler bulk extraction procedures that do not require complex single-cell or single-molecule handling.
3Ease of manufacture
If conventional NGS methods are used for mixture analysis, then cost is reduced, but ability to resolve complex mixtures deteriorates
Solution Approach 1:
The method performs preliminary action by preparing and sequencing both strands of DNA molecules before performing the actual mixture analysis. This preliminary sequencing of both strands allows for error correction and accurate genotype determination in subsequent analysis steps, enabling conventional NGS methods to achieve high-resolution mixture deconvolution that would otherwise require more expensive and complex specialized sequencing systems.
Data Source
AI summary
Methods and associated reagents for assessing and resolving nucleic acid mixtures and/or mixed cell populations are disclosed herein. Some embodiments of the technology are directed to utilizing Duplex Sequencing for assessing and resolving nucleic acid mixtures (e.g., multichimeric mixtures, mixtures of nucleic acids from more than one source, etc.) in a sample and associated applications. Other embodiments are directed to detecting and quantifying a donor source of nucleic acid from a mixture.