Methylated Adapter High-Throughput Sequencing Library Construction
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for detecting DNA methylation in specified genomic regions, such as promoter regions, CpG island regions, and imprinted gene regions, are not sufficiently effective and require improvement.
Innovation Solution
A method for constructing a high-throughput sequencing library involves fragmenting genomic DNA, end-repairing, adding a base A, ligating a methylated adapter, performing hybrid capture with specific probes, PCR amplification, and bisulfite treatment to create a library that can be used for sequencing and analysis of methylation information in specified genomic regions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional methylation detection methods are used, then the detection process is simpler, but the detection accuracy and coverage of methylation sites are insufficient
Solution Approach 1:
The method segments the genome into specific regions of interest (promoter regions, CpG islands, CGI shores, imprinted gene regions) using hybrid capture with region-specific probes. This segmentation allows focused sequencing and analysis on methylation-prone regions, improving detection accuracy while managing complexity through targeted approach
Solution Approach 2:
The method performs preliminary actions including DNA fragmentation, end repair, A-tailing, adapter ligation, and hybrid capture before sequencing. These preliminary steps prepare the DNA library with methylated adapters and region-specific enrichment, ensuring high-quality input for sequencing and improving methylation site identification accuracy
2Quantity of substance
If comprehensive genome coverage is achieved, then more methylation sites are detected, but the detection cost and time increase
Solution Approach 1:
The method applies local quality by using different probes targeted to specific genomic regions with different methylation characteristics (promoters, CpG islands, shores, imprinted regions). Each region receives customized probe coverage, ensuring comprehensive methylation site detection across functionally important regions without requiring uniform whole-genome sequencing
Solution Approach 2:
Hybrid capture with region-specific probes is performed as a preliminary enrichment step before sequencing. This pre-enrichment concentrates the sequencing depth on regions most likely to contain methylation sites, achieving comprehensive coverage of methylation-prone regions while reducing the total sequencing time and cost compared to whole-genome approaches
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 enables accurate and efficient detection of methylation information in specified genomic regions, improving the coverage and accuracy of methylation site identification and providing comprehensive methylation data.
Implementation Method 1
ligating a methylated adapter
Implementation Method 2
performing hybrid capture with specific probes, PCR amplification, and bisulfite treatment
Data Source
AI summary
The present invention provides a method for constructing a high-throughput sequencing library, which comprises: fragmenting genomic DNA; end-repairing the DNA fragments; adding a base A to the 3′ end of the end-repaired DNA fragments; connecting the DNA fragments having cohesive end A with a methylated adapter; carrying out hybrid capture on the connection products by using specific probes to obtain object fragments; treating the object fragments with bisulfite, to convert non-methylated cytosines to uracils; PCR amplifying the converted object fragments; and separating and purifying the amplification products, wherein the amplification products constitute the high-throughput sequencing library. The present invention also provides a method and an apparatus for identifying methylation information in specified genome regions of a sample.