Duplex Sequencing Adapters for Single-End Strand Separation
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Solution Overview
Problem
Existing duplex sequencing methods using asymmetric primer binding sites are prone to errors due to degradation and daisy-chaining, and are limited to paired-end sequencing approaches, necessitating alternative methods for broader application on various sequencing platforms.
Innovation Solution
Introduce asymmetry between DNA strands by creating a nucleotide difference in the adapter sequences or differential labeling, using adapter nucleic acid sequences with primer binding domains, strand defining elements, and single molecule identifiers to separate and sequence both strands independently.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If asymmetric primer binding sites are used in Y-shaped adapters for duplex sequencing, then separate amplification of two DNA strands is achieved, but the free ends of adapters are prone to degradation by exonucleases and daisy-chaining occurs
Solution Approach 1:
The patent applies asymmetry by introducing a non-complementary single-stranded overhang region in the adapter sequence. This asymmetric structure allows one adapter to bind specifically to its complementary strand while preventing binding to the non-complementary strand, thereby enabling strand-specific amplification without the harmful effects of symmetric Y-shaped adapters
Solution Approach 2:
The patent modifies only the local structure of the adapter by adding a single-stranded overhang region with specific nucleotide sequence, while keeping the rest of the adapter structure intact. This localized modification provides strand specificity without requiring the complex Y-shaped structure that is susceptible to degradation
2Reliability
If Y-shaped adapters with asymmetric primer binding sites are used, then duplex sequencing can be performed, but the method is limited to paired-end sequencing approaches
Solution Approach 1:
The patent creates a universal adapter design with a non-complementary overhang that can be used with both paired-end and single-end sequencing platforms. The adapter structure is simplified to work across different sequencing technologies, making the duplex sequencing method broadly applicable without requiring platform-specific optimizations
3Measurement precision
If asymmetric primer binding sites are introduced into adapters, then separate products from two DNA strands are obtained, but the free ends can anneal to other molecules resulting in daisy-chaining
Solution Approach 1:
The patent introduces asymmetry through a non-complementary single-stranded overhang that provides precise strand differentiation. The asymmetric sequence ensures that only the correct complementary strand can bind, preventing the free ends from annealing to non-complementary molecules and thus eliminating daisy-chaining
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 eliminates amplification and sequencing errors, enabling accurate duplex sequencing on single-end sequencing platforms and simplifying broader application across different sequencing technologies.
Implementation Method 1
each adapter nucleic acid sequence includes a primer binding domain, a strand defining element (SDE), a single molecule identifier (SMI) domain, and a ligation domain. The SDE of the first adapter nucleic acid sequence may be at least partially non-complementary to the SDE of the second adapter nucleic acid sequence.
Data Source
AI summary
Disclosed herein are adapter nucleic acid sequences, double-stranded complexed nucleic acids, compositions, and methods for sequencing a double-stranded target nucleic acid with applications to error correction by duplex sequencing.