Modified Transposase Adapters for Low-DNA Sequencing
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Solution Overview
Problem
Current transposase-adapter complexes for DNA fragmentation in next-generation sequencing (NGS) exhibit bias, leading to higher duplication rates and require time-consuming adapter removal, making them inefficient for processing samples with low DNA amounts.
Innovation Solution
Development of isolated synthetic nucleic acid adapters with modifications at the 5' or 3' termini and phosphorothioate bonds to enhance randomness and reduce duplication rates, allowing for direct processing of low-DNA samples without prior adapter removal.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional transposase-adapter complexes are used for DNA fragmentation, then the fragmentation process is efficient in terms of time and labor, but the fragmentation is biased leading to higher duplication rates
Solution Approach 1:
The patent modifies the adapter sequences by changing specific nucleotide positions (particularly positions 1 and 19) to create variant adapters that reduce fragmentation bias. This involves altering the chemical composition and structure of the adapter DNA to optimize transposase binding and cleavage patterns, thereby improving fragmentation randomness while maintaining efficiency
Solution Approach 2:
The patent creates composite adapter structures combining modified recognition sequences with functional regions. These composite adapters integrate multiple elements: transposase binding sites, cleavage sites, and sequencing adapter regions, all optimized together to achieve both high fragmentation efficiency and improved randomness through coordinated design
2Device complexity
If conventional transposase-based NGS sample preparation methods are used, then the process is relatively simple, but adapter removal is required which is time consuming
Solution Approach 1:
The patent extracts the adapter removal step from the preparation workflow by designing adapters that do not require removal. The modified adapters are designed to remain attached to the DNA fragments throughout the sequencing process, eliminating the need for separate removal operations and reducing overall processing time
Solution Approach 2:
The patent designs universal adapters that serve multiple functions simultaneously: they facilitate transposase-mediated fragmentation, provide sequencing priming sites, and enable direct amplification without requiring removal. This multi-functionality consolidates multiple steps into one, eliminating the adapter removal bottleneck
3Adaptability or versatility
If conventional transposase methods are used, then the process is suitable for standard DNA amounts, but it is not suitable for processing samples with very low amount of DNAs
Solution Approach 1:
The patent modifies adapter parameters including sequence composition, length, and chemical modifications to enhance transposase activity and binding affinity. These changes optimize the reaction efficiency for low-DNA inputs, enabling reliable processing of samples with very limited genetic material where conventional methods fail
4Reliability
If transposase-adapter complexes with native recognition sequences are used, then the transposase activity is maintained, but the fragmentation shows bias resulting in more reads in some regions and less in others
Solution Approach 1:
The patent applies local quality changes by modifying specific positions within the adapter sequence (particularly positions 1 and 19) while leaving other regions unchanged. This targeted modification approach locally alters transposase binding characteristics to reduce bias in specific genomic regions while preserving overall transposase activity and function
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
The modified adapters improve the randomness of DNA fragmentation, reduce duplication rates, and enable efficient processing of low-DNA samples, reducing sequencing effort and time, while maintaining transposase activity.
Implementation Method 1
transposase is a class of enzyme that binds to the ends of a transposon and catalyzes the movement of the transposon to another part of the genome by a cut and paste mechanism
Implementation Method 2
isolated synthetic nucleic acid adapters with modifications at the 5' or 3' termini and phosphorothioate bonds
Data Source
Figure 1
Figure 2
Figure 3A~3B
AI summary
The present invention relates to transposase adapters and uses thereof, including uses in preparing DNA molecules, in vitro amplification, sequencing of nucleic acids, and screening of DNA libraries for sequences of interest as well as nucleic acid delivery.