Nucleic Acid Library Preparation via Splint Hybridization
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Solution Overview
Problem
Current methods for preparing nucleic acid sequencing libraries via ligation of adapter sequences are time-consuming and expensive.
Innovation Solution
The method involves combining single-stranded nucleic acid binding protein-bound single-stranded nucleic acid (SSB-bound ssNA) with adapter oligonucleotides and splint oligonucleotides to form complexes, where the splint oligonucleotides are hybridized to the SSB-bound ssNA and adapter oligonucleotides, with adjacent ends being covalently linked for efficient library preparation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If ligation of adapter sequences is used for library preparation, then library preparation can be achieved, but time and cost increase significantly
Solution Approach 1:
The method segments the library preparation process into distinct functional components: SSB-bound ssNA provides template stability, splint oligonucleotides provide hybridization-mediated positioning, and adapter oligonucleotides provide sequencing compatibility. This segmentation allows each component to perform its specific function efficiently without requiring time-consuming ligation steps, thereby resolving the contradiction between achieving library preparation and reducing time loss.
Solution Approach 2:
The splint oligonucleotide acts as an intermediary that hybridizes to both the SSB-bound ssNA and the adapter oligonucleotide, bringing them into proximity and enabling direct attachment. This intermediary mechanism eliminates the need for traditional ligation enzymes and time-consuming ligation reactions, thus improving productivity while reducing time required for library preparation.
2Productivity
If ligation of adapter sequences is used for library preparation, then library preparation can be achieved, but expense increases
Solution Approach 1:
The system employs self-service mechanisms where the splint oligonucleotide autonomously hybridizes to position the adapter, and the SSB-bound ssNA naturally maintains template stability without requiring additional enzymes or reagents. This self-organizing approach eliminates expensive ligation enzymes and reduces reagent costs, thereby improving productivity while reducing the expense of library preparation.
Solution Approach 2:
The method changes the fundamental parameters of the library preparation process by replacing enzymatic ligation with hybridization-based assembly. This parameter change from chemical/enzymatic bonding to physical hybridization bonding significantly reduces reagent costs and simplifies the manufacturing process, resolving the contradiction between achieving library preparation and reducing expense.
3Reliability
If traditional adapter ligation methods are used, then libraries can be prepared, but the process is time-consuming
Solution Approach 1:
The splint oligonucleotide is pre-designed with specific hybridization sequences that complement both the SSB-bound ssNA and the adapter oligonucleotide. This preliminary design ensures that when components are mixed, they automatically self-assemble into the correct configuration through hybridization, eliminating the need for time-consuming ligation reactions while maintaining reliability through predictable and specific base-pairing interactions.
Solution Approach 2:
The method substitutes the mechanical/enzymatic ligation system with a hybridization-based assembly system. Instead of using ligase enzymes to covalently bond adapters to DNA fragments, the system uses complementary base-pairing to bring components together and enable direct attachment. This substitution dramatically reduces process duration while maintaining reliability through the specificity of nucleic acid hybridization.
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 significantly reduces the time and cost required for library preparation, achieving higher efficiency in converting DNA into sequencing libraries compared to existing methods, particularly for degraded and ancient DNA samples.
Implementation Method 1
combining single-stranded nucleic acid binding protein-bound single-stranded nucleic acid (SSB-bound ssNA), an adapter oligonucleotide, and a splint oligonucleotide, to form complexes including the splint oligonucleotide hybridized to a terminal region of the SSB-bound ssNA and to the adapter oligonucleotide
Data Source
AI summary
Provided are methods of producing nucleic acid libraries. The methods include combining single-stranded nucleic acid binding protein-bound single-stranded nucleic acid (SSB-bound ssNA), an adapter oligonucleotide, and a splint oligonucleotide, to form complexes including the splint oligonucleotide hybridized to a terminal region of the SSB-bound ssNA and to the adapter oligonucleotide. An end of the first adapter oligonucleotide is adjacent to an end of the first terminal region of the SSB-bound ssNA, and the methods may further include covalently linking the adjacent ends. Also provided are compositions and kits that find use, e.g., in practicing the methods of the present disclosure.