Vesicular Adaptor for Cyclic Single-Stranded Library Construction
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Solution Overview
Problem
Current nucleic acid sequencing technologies face challenges in constructing high-efficiency cyclic single-stranded libraries, particularly for the Complete Genomics sequencing platform, due to low ligating efficiency and the unsuitability of linear double-stranded libraries.
Innovation Solution
A method using an oligonucleotide vesicular adaptor is developed to construct cyclic single-stranded libraries by ligating the adaptor with a double-stranded DNA fragment, amplifying, isolating single-stranded DNA, and cyclizing it, with specific terminal structures and nucleases to achieve high throughput and accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If a conventional adaptor is used for library construction, then the library can be constructed, but the ligating efficiency is low and many by-products arise
Solution Approach 1:
The adaptor is divided into distinct functional regions: a 5' paired double-stranded region for initial ligation, a 3' paired double-stranded region with overhang for secondary ligation, and a vesicular non-paired region that prevents misligation. This segmentation allows sequential ligation steps that improve efficiency while reducing by-products
Solution Approach 2:
Different regions of the adaptor have specialized structures optimized for specific functions: the 5' region provides stable initial binding, the 3' region with 3' overhang enables directional ligation and prevents self-ligation, and the vesicular region provides structural stability. This local optimization resolves the contradiction between ligation efficiency and by-product formation
2Adaptability or versatility
If a linear double-stranded library is constructed, then it is suitable for mainstream sequencing platforms, but it is not suitable for the CG sequencing platform which requires cyclic single-stranded libraries
Solution Approach 1:
The library structure is transformed from static linear double-stranded to dynamic cyclic single-stranded form through a multi-step process: initial ligation creates linear structures, PCR amplification generates double-stranded products, denaturation produces single-stranded DNA, and final cyclization creates the required cyclic structure. This dynamic transformation enables platform-specific optimization
Solution Approach 2:
The library undergoes parameter changes including strand number (double to single), topology (linear to cyclic), and structural configuration. These parameter transformations, driven by the adaptor's specific structure and controlled by pH, temperature, and enzyme conditions, enable conversion to the CG-platform required format while maintaining sequencing suitability
3Ease of manufacture
If the 3' paired double-stranded region has both overhang and phosphorylation, then sticky terminal is provided for ligation, but the structure becomes more complex
Solution Approach 1:
The 3' paired double-stranded region merges multiple functions into a single structural element: it provides the sticky terminal through 3' overhang for ligation capability, incorporates 5' phosphorylation for bond formation, and maintains double-stranded stability. This merging achieves enhanced ligation capability without proportionally increasing overall complexity
Solution Approach 2:
The 3' paired double-stranded region serves multiple purposes: it provides the sticky end for ligation, ensures directional orientation through asymmetry, prevents adaptor self-ligation, and facilitates stable binding to the DNA fragment. This multi-functionality justifies the localized structural complexity while improving overall ligation ease
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 method provides a high-quality nucleic acid sequencing library with improved ligating efficiency and accuracy, suitable for high-throughput sequencing platforms like Complete Genomics, offering high stability, repeatability, and reliable sequencing data.
Implementation Method 1
ligating the oligonucleotide vesicular adaptor with a double-stranded DNA fragment to provide a ligation product
Implementation Method 2
subjecting the double-stranded structure obtained in (a) to amplification under a pair of primers so as to obtain an amplified double-stranded DNA product
Implementation Method 3
isolating a single-stranded DNA from the amplified double-stranded DNA product obtained in (b)
Implementation Method 4
subjecting the single-stranded DNA obtained in (c) to cyclization in the presence of a cycling single-stranded molecule, so as to obtain the cyclic single-stranded library
Implementation Method 5
treating the cyclic single-stranded library with an exonuclease I and an exonuclease III
Data Source
AI summary
Provided are a vesicular adaptor and a single-chain cyclic library constructed by using the adaptor. The library can be used for RNA sequencing and other sequencing platforms dependent on a single-stranded cyclic library, and has the advantage of high throughput sequencing, high accuracy and simple operations.