Sequencing Library Construction via Probe Hybridization and Cleavage
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Solution Overview
Problem
Current high-throughput sequencing methods face challenges with computational assembly errors due to random fragmentation of genomic DNA, particularly in organisms with large genome sizes, and are resource-intensive, limiting their capacity for accurate genome assembly and detection of rearrangements and duplications.
Innovation Solution
The construction of sequencing libraries involves generating adaptor-ligated duplexes of oligonucleotide probe-hybridized template sequence fragments with chemically-active groups for efficient end cleavage and ligation, allowing for library construction in a single reaction tube with reduced steps and no purification requirements, optimizing DNA fragmentation and hybridization conditions for improved sequencing efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If random fragmentation of genomic DNA is used for high throughput sequencing, then sequencing throughput is improved, but computational assembly errors increase
Solution Approach 1:
The patent applies preliminary action by performing targeted fragmentation and end preparation steps before sequencing. Specifically, the method includes selective fragmentation of genomic DNA at predetermined locations and preparation of fragment ends with specific chemical groups, ensuring that fragments are ready for efficient adapter ligation and sequencing while maintaining assembly accuracy through controlled fragmentation rather than random breaking.
2Manufacturing precision
If multiple steps of library construction are performed separately, then library construction accuracy is improved, but time and reagent consumption increase
Solution Approach 1:
The patent merges multiple library construction steps into a single integrated reaction. Specifically, it combines fragmentation, end preparation, adapter ligation, and fragment purification into one streamlined protocol that can be completed in a single reaction tube, significantly reducing time and reagent consumption while maintaining construction accuracy through optimized reaction conditions.
Solution Approach 2:
The patent employs universal reagents and reaction conditions that serve multiple functions simultaneously. The reaction system uses a single set of reagents that perform fragmentation, end modification, and adapter ligation functions, eliminating the need for separate reagent sets for each step and thereby reducing overall reagent consumption and procedural complexity.
3Reliability
If conventional library construction methods are used, then sequencing reliability is maintained, but resource consumption increases
Solution Approach 1:
The patent optimizes reaction parameters to improve efficiency. It uses specific temperature conditions, pH values, and enzyme concentrations that enhance the efficiency of fragmentation and ligation reactions, thereby reducing the amount of reagents needed while maintaining sequencing reliability. The method also employs optimized fragment size distributions that reduce waste and improve sequencing success rates.
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 time and reagent costs, enhances sequencing accuracy by minimizing computational assembly errors, and improves the capacity for genome assembly and detection of complex rearrangements and duplications, making it suitable for next-generation sequencing applications.
Implementation Method 1
generating adaptor-ligated duplexes of oligonucleotide probe-hybridized template sequence fragments
Implementation Method 2
Probes of the invention may include a chemically-active group at their 5′ or 3′ ends, or both, to facilitate the cleavage of their 5′ or 3′ ends, or both, following their hybridization
Implementation Method 3
Adaptors, specific to these ends, are ligated to the hybridized probe/template fragments
Data Source
AI summary
Rapid methods, capable of being performed in a single reaction tube, are described herein for constructing libraries for high-throughput polynucleotide sequencing applications, such as next generation sequencing (NGS) applications. Oligonucleotide probes include chemically-active groups at their 5′ or 3′ ends, or both, to facilitate the cleavage of their 5′ or 3′ ends, or both, following their hybridization to the single-stranded ends of frayed template fragments. Cleavage of probe ends reveal single-stranded regions at the ends of the hybridized fragments. Adaptors, specific to these ends, are ligated to the hybridized probe/template fragments, and blunt end fragments are ligated to blunt ends of hybridized probe/template fragments, if present, to generate the adaptor-ligated fragments of the library.