Single-Stranded Splint Circularization for Sequencing Libraries
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Solution Overview
Problem
Existing nucleic acid library preparation methods face challenges in efficiently forming covalently closed circular molecules for downstream amplification and sequencing workflows, particularly due to the lack of effective methods for circularizing linear library molecules.
Innovation Solution
The use of single-stranded splint strands that hybridize with linear library molecules to form library-splint complexes with a nick, which can be ligated to create covalently closed circular molecules, facilitating downstream amplification and sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If linear library molecules are used directly in downstream workflows, then the process is simpler, but amplification efficiency and sequencing accuracy are reduced
Solution Approach 1:
A splint oligonucleotide serves as an intermediary molecule that hybridizes to both ends of the linear library molecule, bringing them into proximity and enabling ligation to form a circular structure. This mediator facilitates the circularization process without requiring complex enzymatic machinery or multiple steps, thus improving amplification efficiency while keeping the process relatively simple.
Solution Approach 2:
The splint oligonucleotide is designed with predetermined hybridization sequences that complementary match the library molecule ends. By performing the hybridization and circularization steps before downstream amplification and sequencing, the library molecules are pre-prepared in the optimal circular form, eliminating the need for complex in-situ circularization during the main workflow.
2Measurement precision
If covalently closed circular molecules are formed, then sequencing accuracy is improved, but the ligation step adds process complexity
Solution Approach 1:
The splint oligonucleotide acts as a template and mediator during the ligation reaction, guiding the joining of library molecule ends to form covalently closed circular structures. This intermediary approach ensures high sequencing accuracy by creating stable, properly structured circular molecules, while the splint can be easily removed afterward, minimizing the impact on overall process simplicity.
Solution Approach 2:
The splint oligonucleotide is a temporary component used during circularization that can be discarded (removed) after serving its purpose. Following ligation, the splint is no longer needed and can be eliminated through simple denaturation or purification steps, allowing the library molecules to proceed to sequencing without carrying unnecessary complexity.
3Stability of the object's composition
If splint strands are used to circularize library molecules, then molecular stability is improved, but additional reagents and steps are required
Solution Approach 1:
The splint oligonucleotide provides structural stability during the circularization process by hybridizing to both ends of the library molecule and maintaining them in a fixed orientation. This intermediary structure ensures that the library molecule forms a stable covalently closed circular conformation, while the splint itself can be designed with simple sequences that do not complicate the overall reaction system.
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 enables efficient formation of covalently closed circular molecules, enhancing the efficacy of downstream amplification and sequencing processes by stabilizing the library molecules and improving sequencing accuracy.
Implementation Method 1
The single-stranded splint strands can hybridize to portions of library molecules to form library-splint complexes
Data Source
AI summary
The present disclosure provides compositions comprising nucleic acid single-stranded splint strands, including kits, and methods that employ the single-stranded splint strands. The single-stranded splint strands can hybridize to portions of linear library molecules to form circularized library-splint complexes having a nick, where the nick can be ligated to form covalently closed circular molecules which can be subjected to downstream amplification and sequencing workflows.