Sequencing Library With Circularized Adaptor for Error-Free Amplification
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Solution Overview
Problem
Current next-generation sequencing technologies suffer from high error rates, particularly due to PCR amplification errors, which hinder accurate DNA sequencing, especially in detecting low-frequency mutations and achieving comprehensive genome coverage.
Innovation Solution
A sequencing library is developed using a method that involves ligating a target sequence with an adaptor sequence containing a nicking site or gap, followed by circularization and strand displacement amplification, allowing for independent replication of target sequences and effective error removal, resulting in accurate and uniform genome sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If PCR amplification is used during library preparation, then the DNA quantity is increased, but sequencing errors are introduced and accuracy is reduced
Solution Approach 1:
The patent extracts and removes the harmful PCR amplification step from the library preparation process. By using isothermal amplification instead of PCR, the method eliminates PCR-specific errors while maintaining DNA quantity increase, thus resolving the contradiction between quantity enhancement and accuracy preservation
Solution Approach 2:
The patent changes the amplification parameters by switching from thermal cycling (PCR) to isothermal conditions. This parameter change eliminates the denaturation/annealing/extension cycles that cause PCR errors, while still achieving sufficient DNA amplification for sequencing, thereby maintaining both quantity and accuracy
2Measurement precision
If tagging methods are used to correct errors, then sequencing accuracy is improved, but the method can only target small genomes or few genes and cannot achieve complete genome detection
Solution Approach 1:
The patent creates a universal library preparation method that can be applied to any genome size or target region. By eliminating PCR and using isothermal amplification with random priming, the method achieves both high accuracy and complete genome coverage, making it adaptable to whole-genome sequencing, targeted sequencing, and metagenomics without limitation
Solution Approach 2:
The patent uses random priming to segment the genome into multiple starting points for amplification. This segmentation approach allows comprehensive coverage of the entire genome while maintaining accuracy, as each segment is independently amplified without PCR errors and can be assembled into complete genomic information
3Quantity of substance
If rolling circle amplification is used, then DNA amplification is achieved, but severe amplification bias occurs and uniform genome coverage is difficult to obtain
Solution Approach 1:
The patent inverts the approach by using isothermal amplification without circularization. Instead of rolling circle amplification that starts from circular templates and creates bias, the method uses linear isothermal amplification from random priming sites, which eliminates the inherent bias of rolling circle amplification while maintaining amplification efficiency
Solution Approach 2:
The patent uses excessive random priming to ensure every region of the genome is targeted. By providing numerous random priming sites throughout the genome and using isothermal amplification, the method achieves uniform coverage without the bias of rolling circle amplification, as multiple overlapping amplifications ensure even difficult-to-amplify regions are covered
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 sequencing errors, achieves uniform genome amplification, and enables precise detection of mutations, even at low frequencies, compatible with various sequencing platforms and suitable for both whole-genome and targeted sequencing applications.
Implementation Method 1
The adaptor sequence is ligated to the target sequence
Implementation Method 2
followed by circularization and strand displacement amplification, allowing for independent replication of target sequences
Data Source
AI summary
The present invention discloses a sequencing library comprising a nucleotide sequence. The sequence comprises a linker sequence and two target sequences. Two ends of the linker sequence are respectively linked to the target sequences and the two target sequences are direct repeat sequences. The present invention further discloses preparation and use of the sequencing library. The present invention overcomes the high error rate problem of current DNA sequencing technologies, especially in a way of very low coverage bias, and can be used to detect low frequency mutations in different kinds of samples.