Nucleic Acid Library Preparation via Oligonucleotide Pool Hybridization
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Solution Overview
Problem
High-throughput sequencing technologies face limitations in library preparation costs and throughput, which hinder efficient nucleic acid analysis, particularly in determining overhang lengths and sequences for accurate nucleic acid library generation.
Innovation Solution
A method involving the combination of target nucleic acids with an oligonucleotide pool, where oligonucleotides have complementarity regions capable of hybridizing to overhangs, allowing for hybridization products to be formed, enabling efficient nucleic acid library production and sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional library preparation methods are used, then sequencing can be performed, but library preparation costs and throughput are limiting factors
Solution Approach 1:
The patent segments the library preparation process into two distinct stages: (1) a hybridization stage where oligonucleotides with complementarity regions bind to target nucleic acid overhangs, and (2) a ligation stage where adapters are joined to the hybridized complexes. This segmentation enables parallel processing and simplifies each individual step, thereby increasing overall throughput while reducing complexity.
Solution Approach 2:
The patent introduces oligonucleotide complexes as intermediary structures that facilitate library preparation. These complexes form between oligonucleotides and target nucleic acids during hybridization, serving as mediators that enable subsequent adapter ligation. This intermediary approach simplifies the overall process by providing a stable platform for efficient adapter attachment.
2Measurement precision
If accurate overhang length determination is performed, then sequencing accuracy improves, but library preparation time increases
Solution Approach 1:
The patent performs preliminary actions by first hybridizing oligonucleotides to target nucleic acids and forming stable complexes before performing ligation. This preliminary hybridization step pre-positions the adapters for efficient subsequent ligation, reducing overall preparation time while maintaining accurate overhang length determination through the use of complementarity region identification sequences.
Solution Approach 2:
The patent utilizes parameter changes in the form of temperature-dependent hybridization conditions to optimize both accuracy and speed. By controlling hybridization temperature and other parameters, the method achieves specific binding of oligonucleotides to their complementary overhangs, enabling accurate length determination without excessive time consumption.
3Reliability
If oligonucleotides with varying complementarity region lengths are used, then hybridization specificity improves, but oligonucleotide pool complexity increases
Solution Approach 1:
The patent segments the oligonucleotide pool into distinct groups based on complementarity region length, with each group containing oligonucleotides designed for specific overhang lengths. This segmentation allows for systematic management of pool complexity while maintaining high hybridization specificity through the use of length-matched complementarity regions.
Solution Approach 2:
The patent applies local quality by designing oligonucleotides with specific complementarity region lengths tailored to match particular overhang lengths in the target nucleic acids. Each local region of the oligonucleotide pool is optimized for specific hybridization tasks, improving overall reliability while managing complexity through localized optimization rather than uniform 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
This approach enhances the accuracy and efficiency of nucleic acid library generation by specifically hybridizing oligonucleotides to target nucleic acids based on overhang lengths and sequences, facilitating effective sequencing and analysis.
Implementation Method 1
Each oligonucleotide of the oligonucleotide pool may include (i) a complementarity region capable of hybridizing to an overhang in a target nucleic acid
Data Source
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AI summary
Provided are methods of producing nucleic acid libraries. In certain aspects, the methods include combining target nucleic acids (e.g., 5' phosphorylated nucleic acids) and an oligonucleotide pool. Oligonucleotides of the oligonucleotide pool may include complementarity regions of varying length and nucleotide sequence, and a complementarity region identification sequence. In such aspects, the combining is under conditions in which oligonucleotides of the oligonucleotide pool hybridize to nucleic acids of the target nucleic acids (e.g., 5' phosphorylated nucleic acids) having overhang regions that are complementary in sequence and have corresponding lengths with respect to the complementarity regions of the oligonucleotides. Compositions and kits that find use, e.g., in practicing the methods of the present disclosure are also provided.