Crowding-Agent Hybridization Buffer for NGS Off-Target Reduction
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Solution Overview
Problem
Classic library preparation and enrichment methods for next-generation sequencing (NGS) are time-intensive and throughput-limiting, leading to inefficiencies in nucleic acid selection.
Innovation Solution
The use of a hybridization buffer comprising a crowding agent, human Cot-1 DNA, a destabilizing agent, and blockers, including oligonucleotides that bind to adaptors with specific sequences, to prevent off-target hybridization and enhance nucleic acid selection efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If classic library preparation and enrichment methods are used, then sequencing can be performed, but the process is time-intensive and results in throughput-limiting bottlenecks
Solution Approach 1:
The patent modifies the hybridization conditions by introducing a destabilizing agent that prevents G-quadruplex formation, changing the physical-chemical parameters of the hybridization buffer to improve off-target capture and increase sequencing throughput
Solution Approach 2:
The patent introduces an intermediary substance (destabilizing agent) that mediates between the probe and target nucleic acids by preventing unwanted secondary structures, thereby improving hybridization efficiency and reducing off-target capture
2Measurement precision
If classic enrichment methods are used, then nucleic acid selection can be performed, but off-target capture occurs reducing accuracy
Solution Approach 1:
The patent changes the chemical composition of the hybridization buffer by adding a destabilizing agent that specifically prevents G-quadruplex formation, thereby improving the specificity of probe-target hybridization and reducing off-target capture
Solution Approach 2:
The patent converts the potential harm of G-quadruplex formation (which causes off-target capture) into a benefit by using a destabilizing agent to prevent it, thereby improving enrichment accuracy
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
Improves the efficiency of nucleic acid selection by reducing off-target capture, thereby enhancing the overall throughput and accuracy of sequencing processes.
Implementation Method 1
a hybridization buffer including a crowding agent and at least one of human Cot-1 DNA
Implementation Method 2
a hybridization buffer including a crowding agent
Implementation Method 3
a blocker including an oligonucleotide, wherein the blocker is capable of binding to an adaptor
Data Source
AI summary
This disclosure describes a hybridization buffer including a crowding agent, a method that includes using the hybridization buffer, and a kit including the hybridization buffer. This disclosure also describes blockers for use in hybrid capture methods, methods of using those blockers, and a kit including those blockers. Additionally, this disclosure describes a method of hybrid capture that does not include amplifying the library members using PCR prior to sequencing the library members.