Sequencing Library Construction from Trace DNA via Single-Step Tagmentation
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Solution Overview
Problem
Current methods for constructing sequencing libraries for second-generation sequencing technologies are complex and result in significant sample loss due to multiple purification steps, making them unsuitable for trace or scarce samples.
Innovation Solution
A method and apparatus for constructing sequencing libraries using single-stranded DNA molecules, which involves forming a poly(C) tail at the 3′-terminus, using an extension primer with a Poly(G) unit, ligating an adapter, and amplifying the library, minimizing purification steps and sample loss.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional library construction methods (Trueseq and Nextera systems) are used, then sequencing libraries can be constructed, but multiple purification steps are required causing significant sample loss and making them unsuitable for trace samples
Solution Approach 1:
The invention extracts and eliminates the multiple purification steps from the traditional library construction workflow. By using a single-step tagmentation reaction that simultaneously fragments DNA, adds adapters, and prepares libraries for sequencing, the method removes the need for separate purification operations between each step, thereby minimizing sample loss while simplifying the overall procedure
Solution Approach 2:
The invention merges multiple separate operations (DNA fragmentation, adapter ligation, and library preparation) into a single tagmentation reaction step. This consolidation eliminates the need for intermediate purification steps, reducing both operational complexity and sample loss, while maintaining the ability to construct high-quality sequencing libraries from trace samples
2Quantity of substance
If multiple purification steps are performed, then library construction can be completed, but large beginning amount of DNA is required and huge amount of information is lost
Solution Approach 1:
The invention converts the limitation of having only trace amounts of DNA into an advantage by designing a method that works specifically well with minimal input material. The single-step tagmentation approach is optimized to efficiently process nanogram-level samples, converting what was previously a constraint (small sample size) into a beneficial feature that reduces the risk of contamination and degradation associated with handling larger sample volumes
3Ease of operation
If traditional library construction methods are used, then sequencing libraries can be constructed, but purified operation is required at every step increasing operation complexity
Solution Approach 1:
The invention performs preliminary actions by incorporating all necessary components (adapters, fragmentation enzymes, and ligation machinery) into a single tagmentation reaction mixture before the reaction begins. This pre-preparation allows the entire library construction process to proceed in one step without intermediate purification, reducing both operational complexity and time while ensuring all necessary transformations occur simultaneously
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 allows for efficient and sensitive construction of sequencing libraries from trace samples with reduced DNA loss, maintaining specificity and integrity of gene information, and is applicable in high-throughput sequencing.
Implementation Method 1
fulfill complementary condition and extension reaction utilizing DNA 3′-terminus Poly(C)n and extension primer containing Poly(G)m
Data Source
AI summary
A method for constructing a sequencing library based on a single-stranded DNA molecule is provided comprising: (1) forming a poly(C)n tail at a 3'-terminus of the single-stranded DNA molecule, to obtain a single-stranded DNA molecule with the poly(C)n tail with n representing a number of base C, and n being an integer ranging from 5 to 30; (2) obtaining a double-stranded DNA molecule by using an extension primer based on the single-stranded DNA molecule with the poly(C)n tail, with the extension primer comprising a H(G)m unit at a 3'-terminus thereof, H being base A, base T or base C, m being a number of base G, and m being an integer ranging from 5 to 15; and (3) ligating an adapter to one terminus of the double-stranded DNA molecule remote from the H(G)m unit, and amplifying the resulting ligation product to obtain an amplification product forming the sequencing library.