Stem-Loop Adaptor for Strand-Specific DNA Sequencing
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Solution Overview
Problem
Existing nucleic acid sequencing methods face challenges in efficiently preparing libraries that allow for separate sequencing of each strand of target nucleic acids, especially for long target molecules, due to the presence of linear byproducts that impede sequencing performance.
Innovation Solution
A method involving the use of stem-loop adaptors with strand cleavage sites, where both ends of a double-stranded target nucleic acid are joined to adaptors, followed by exonuclease treatment to enrich for doubly-adapted molecules, and subsequent cleavage to generate extendable termini for separate sequencing of each strand.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If linear nucleic acid libraries are prepared for sequencing, then library preparation is straightforward, but linear nucleic acid byproducts impede sequencing performance
Solution Approach 1:
The adaptor is segmented into distinct functional regions: a double-stranded stem containing the cleavage site and a single-stranded loop containing the primer binding site. This segmentation allows the cleavage enzyme to specifically access and cleave at the stem region while the loop region remains protected, enabling selective strand separation without affecting the overall adaptor structure or target DNA integrity.
Solution Approach 2:
The stem-loop adaptor structure serves as an intermediary mechanism between the double-stranded target DNA and the sequencing process. The adaptor's unique structure with exposed single-stranded regions allows it to mediate the interaction between the target DNA and sequencing enzymes, facilitating controlled strand separation and enabling separate sequencing of each strand while maintaining library preparation efficiency.
2Productivity
If circular double stranded templates are used for sequencing, then both strands can be read multiple times in contiguous polymerase reads, but separate sequencing of each strand is not achieved
Solution Approach 1:
The cleavage site is pre-positioned within the adaptor stem region before sequencing begins. This preliminary arrangement of the cleavage site allows the cleavage enzyme to selectively separate the strands during the sequencing process, enabling each strand to be sequenced independently while maintaining high throughput by avoiding the need for separate library preparations for each strand.
Solution Approach 2:
The adaptor exhibits local quality differences with a double-stranded stem region containing the cleavage site and a single-stranded loop region containing the primer binding site. This local structural variation allows different regions of the adaptor to perform different functions: the stem region facilitates controlled strand separation while the loop region enables primer binding and extension, thereby achieving both high productivity and strand-specific information retention.
3Loss of information
If adaptors with cleavage sites are used to enable separate strand sequencing, then strand-specific sequencing is achieved, but library preparation complexity increases
Solution Approach 1:
The adaptor adopts a stem-loop (hairpin) structure that introduces curvature and three-dimensional folding to an otherwise linear nucleic acid sequence. This curved configuration allows the double-stranded stem and single-stranded loop to coexist in a compact structure, enabling the cleavage site to be positioned in the stem while the primer binding site remains accessible in the loop, thereby achieving strand-specific sequencing without excessive complexity.
Solution Approach 2:
The adaptor structure utilizes parameter changes in nucleic acid secondary structure, transitioning from a linear single-stranded form to a folded stem-loop structure with distinct double-stranded and single-stranded regions. This parameter change in structural organization allows the cleavage site and primer binding site to be spatially separated while remaining part of a single adaptor molecule, achieving the desired functionality with minimal added complexity.
4Manufacturing precision
If exonuclease treatment is applied to enrich doubly-adapted molecules, then library enrichment efficiency improves, but processing time increases
Solution Approach 1:
The exonuclease treatment selectively extracts or removes singly-adapted molecules and unligated adaptors from the library by degrading their exposed single-stranded ends. This extraction process enriches the library for doubly-adapted circular molecules that lack exposed ends, thereby improving manufacturing precision. The stem-loop structure of the adaptor facilitates this by providing a distinctive topology that allows exonucleases to selectively act on incomplete adaptors.
Solution Approach 2:
The stem-loop adaptor structure provides self-service functionality where the ligated adaptor's own topology (circularized with no free ends) protects it from exonuclease degradation, while unligated or singly-ligated adaptors with free ends are automatically degraded. This self-distinguishing property enables automatic enrichment of correctly ligated products without requiring complex external selection mechanisms, reducing processing time while maintaining high enrichment efficiency.
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 method enables efficient generation of highly enriched libraries, allowing for long single-molecule sequencing without strand orientation bias, and is compatible with various types of target nucleic acids, including genomic DNA and amplification products.
Implementation Method 1
contacting the reaction mixture with an exonuclease thereby enriching for the doubly-adapted target nucleic acid
Implementation Method 2
contacting the reaction mixture with a cleavage agent to cleave the doubly-adapted target nucleic acid at the cleavage sites
Implementation Method 3
extending the extendable termini thereby separately sequencing each strand of the target nucleic acid
Data Source
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AI summary
The invention is a novel method of separately sequencing each strand of a nucleic acid involving the use of an adaptor comprising a strand cleavage site or a strand synthesis termination site. The adaptor may also be self-priming at the strand cleavage site.