Duplex Nucleic Acid Enrichment for Error-Corrected Sequencing
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Solution Overview
Problem
Current genetic analysis methods, such as PCR-CE and MPS, face challenges in distinguishing stutter alleles from genuine alleles, especially in mixed DNA samples, and suffer from PCR errors and coverage imbalance, limiting their utility in forensic and clinical applications.
Innovation Solution
A method involving double-stranded nucleic acid molecules with unique adapters and single molecule identifiers, allowing for separate amplification and sequencing of each strand, followed by comparison to correct errors and generate accurate sequence reads.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If multiplex PCR is used to provide enough DNA for sequencing, then sufficient DNA quantity is achieved, but coverage imbalance and bias toward smaller fragments occur
Solution Approach 1:
The patent divides the amplification process into separate single-strand PCR reactions instead of using multiplex PCR. Each strand is amplified independently with its own optimized primer, eliminating the coverage imbalance that occurs when multiple amplicons of different sizes are amplified simultaneously in multiplex PCR.
2Productivity
If standard MPS is used for sequencing, then high throughput is achieved, but PCR errors and stutter artifacts reduce accuracy
Solution Approach 1:
The patent implements a feedback mechanism by sequencing both strands of the DNA molecule and using the complementary strand's sequence to correct errors in the primary strand. The consensus sequence is generated by comparing both strands, eliminating PCR errors and stutter artifacts while maintaining high throughput.
Solution Approach 2:
The patent creates a complementary copy of the DNA molecule and sequences both the original and the copy. By comparing the two sequences, errors introduced during PCR amplification can be identified and corrected, significantly improving sequence accuracy while maintaining high throughput sequencing capability.
3Quantity of substance
If PCR amplification is performed on degraded DNA, then sufficient template quantity is obtained, but PCR errors and stutter increase
Solution Approach 1:
By sequencing both strands and using the complementary strand as a reference, the method can detect and correct PCR errors that occur during amplification of degraded DNA. This feedback mechanism maintains allele discrimination accuracy even when amplifying damaged templates.
Solution Approach 2:
The patent performs end-repair and adapter ligation on the degraded DNA fragments before PCR amplification. This preliminary processing creates stable, full-length molecules with defined ends, reducing the variability and errors that occur during subsequent PCR amplification of degraded templates.
4Measurement precision
If single-strand consensus sequencing is used, then some error correction is achieved, but duplex sequencing provides superior accuracy
Solution Approach 1:
The patent introduces asymmetry by treating the two DNA strands differently during library preparation - each strand receives a unique adapter configuration. This asymmetric treatment allows the sequencing system to distinguish between forward and reverse strands, enabling more effective error correction through duplex sequencing while managing complexity through systematic design.
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
Enables high-accuracy, cost-effective sequencing of small nucleic acid samples with improved discrimination between alleles, even in degraded samples, and detects low-frequency mutations with high confidence.
Implementation Method 1
amplifying the nucleic acid material; amplifying the first strand in the first sample using at least one single-stranded oligonucleotide at least partially complementary to a sequence in the first adapter sequence
Implementation Method 2
comparing the sequence of the first nucleic acid product to the sequence of the second nucleic acid product; generating an error-corrected sequence read
Data Source
Figure 1A~1C
Figure 2
Figure 3A~3B
AI summary
The present technology relates generally to methods and compositions for targeted nucleic acid sequence enrichment, as well as uses of such enrichment for error-corrected nucleic acid sequencing applications. In some embodiments, highly accurate, error corrected and massively parallel sequencing of nucleic acid material is possible using a combination of uniquely labeled strands in a double-stranded nucleic acid complex in such a way that each strand can be informatically related to its complementary strand, but also distinguished from it following sequencing of each strand or an amplified product derived therefrom. In various embodiments, this information can be used for the purpose of error correction of the determined sequence.