Nucleic Acid Barcoding via Adaptor Ligation for High-Throughput Sequencing
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Solution Overview
Problem
Current methods for detecting and analyzing specific nucleic acid sequences in multiple samples simultaneously are inefficient, particularly in high-throughput assays and sequencing applications, as they lack effective strategies for tagging and identifying target nucleic acids in a cost-effective and scalable manner.
Innovation Solution
The method involves adding adaptor molecules with sticky ends to target nucleic acids, followed by annealing, gap-filling, and ligation to produce adaptor-modified molecules, and employing combinatorial tagging and barcoding techniques using multiple primers to create unique barcode combinations on target nucleotide sequences, enabling efficient identification and analysis of multiple samples in a single assay.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiple target nucleic acids are detected and analyzed simultaneously in multiple samples, then the throughput and efficiency of detection are improved, but the complexity of the assay and cost increase
Solution Approach 1:
The assay is divided into multiple independent reaction mixtures, each containing unique primer pairs and barcode sequences. Each reaction mixture can independently detect specific target nucleic acids, allowing parallel processing of multiple samples while maintaining manageable complexity through modular design
Solution Approach 2:
Universal adaptor molecules are designed that can be used across all reaction mixtures and samples. These adaptors contain common sequencing primer binding sites and barcode regions, enabling a single assay platform to handle multiple samples and targets simultaneously without requiring sample-specific reagent sets
2Loss of time
If multiple samples are analyzed simultaneously, then the time required for detection is reduced, but the accuracy of identifying specific nucleic acid sequences decreases
Solution Approach 1:
Unique barcode nucleotide sequences serve as intermediary identifiers that link each target nucleic acid to its specific reaction mixture and sample origin. These barcodes are incorporated into the amplification products and can be read during sequencing to accurately trace each sequence back to its source sample, maintaining identification accuracy even when multiple samples are processed together
Solution Approach 2:
Each reaction mixture is assigned unique local characteristics including specific barcode sequences and primer combinations. This local differentiation allows the system to distinguish between samples and targets within the pooled reaction, ensuring accurate identification while enabling simultaneous processing
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 efficiency of nucleic acid detection and sequencing by allowing for the simultaneous analysis of multiple samples, reduces costs, and improves the accuracy of identifying specific nucleic acid sequences through unique barcode combinations, facilitating high-throughput applications.
Implementation Method 1
The method entails annealing adaptor molecules to the sticky ends of double-stranded target nucleic acid molecules to produce annealed adaptor-target nucleic acid molecules
Data Source
AI summary
Described herein are methods useful for incorporating one or more adaptors and/or nucleotide tag(s) and/or barcode nucleotide sequence(s) one, or typically more, target nucleotide sequences. In particular embodiments, nucleic acid fragments having adaptors, e.g., suitable for use in high-throughput DNA sequencing are generated. In other embodiments, information about a reaction mixture is encoded into a reaction product. Also described herein are methods and kits useful for amplifying one or more target nucleic acids in preparation for applications such as bidirectional nucleic acid sequencing. In particular embodiments, methods of the invention entail additionally carrying out bidirectional DNA sequencing. Also described herein are methods for encoding and detecting and/or quantifying alleles by primer extension.