Methylated DNA Sequencing via C0T-1 Repetitive Sequence Removal
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Solution Overview
Problem
Current high-throughput sequencing methods for DNA methylation, such as shotgun bisulfite sequencing, MeDIP sequencing, and enzymatic digestion-bisulfite sequencing, face challenges including high sequencing costs, complex data analysis, and the inability to effectively remove repetitive sequences, which leads to increased costs and reduced biological relevance of sequencing data.
Innovation Solution
A method combining methylated DNA immunoprecipitation (MeDIP) with the removal of repetitive sequences using C0T-1 DNA and bisulfite treatment, followed by the rational design of auxiliary adapters and primers to facilitate sequencing, reduces the size of the sequencing library and decreases data analysis complexity, thereby lowering costs and improving the detection of methylation profiles in functional regions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If shotgun bisulfite sequencing is used to achieve high-throughput scanning of DNA methylation patterns, then sequencing throughput is improved, but sequencing cost and data analysis complexity increase significantly
Solution Approach 1:
The patent extracts and removes repetitive sequences from the sequencing library before high-throughput sequencing. By using C0T-1 DNA to specifically bind and remove repetitive elements, the method reduces the proportion of redundant data, thereby decreasing data analysis complexity while maintaining sequencing throughput for detecting methylation patterns in functional regions.
Solution Approach 2:
The patent segments the genomic DNA into functional regions and repetitive sequences through selective enrichment and removal processes. By separating these components and focusing sequencing on functional regions only, the method reduces the overall data volume and analysis complexity while preserving the ability to detect methylation patterns at high throughput.
2Measurement precision
If repetitive sequences are not removed before sequencing, then sequencing coverage is improved, but sequencing cost increases and biological relevance of data decreases
Solution Approach 1:
The patent extracts and removes repetitive sequences from the DNA sample before sequencing using C0T-1 DNA hybridization. This extraction process eliminates redundant data that would otherwise increase sequencing cost and reduce the biological relevance of the data, while maintaining sufficient coverage of functional regions for precise methylation detection.
Solution Approach 2:
The patent applies selective removal of repetitive sequences while preserving functional regions. By locally targeting repetitive elements for removal while maintaining intact the functional genomic regions, the method optimizes the sequencing data to have higher biological relevance and reduces the overall data volume without compromising methylation detection precision in important regions.
3Measurement precision
If MeDIP sequencing is used to enrich methylated DNA, then methylation detection specificity is improved, but repetitive sequences remain in the library increasing sequencing cost
Solution Approach 1:
The patent merges two enrichment strategies: MeDIP for enriching methylated DNA and C0T-1 DNA for removing repetitive sequences. By combining these two approaches in a sequential workflow, the method achieves both high methylation detection specificity and reduced library complexity, eliminating the need for repetitive sequences while maintaining enriched methylated DNA for sequencing.
Solution Approach 2:
The patent performs preliminary removal of repetitive sequences using C0T-1 DNA before performing MeDIP enrichment and sequencing. This preliminary action reduces the complexity of the sequencing library by eliminating repetitive elements that would otherwise remain in the library, thereby reducing sequencing cost while maintaining the specificity of methylation detection through the subsequent MeDIP step.
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 costs and data analysis complexity, allowing for more efficient detection of methylation profiles in functional genomic regions with higher specificity and reduced repetitive sequence interference, achieving a cost-effective and high-throughput sequencing method.
Implementation Method 1
methylated DNA immunoprecipitation (MeDIP)... 5-methylcytosine antibody can be used for immuneprecipitating enriched methylated DNA fragments with high specificity
Implementation Method 2
removal of repetitive sequences using C0T-1 DNA
Implementation Method 3
C0T-1 DNA is labeled with biotin. Magnetic beads coated with avidin... the repetitive sequences are hybridized with the C0T-1 DNA labeled with biotin to obtain a complex containing the repetitive sequences—C0T-1 DNA labeled with biotin—magnetic beads coated with avidin. The magnetic bead complexes are separated and discarded.
Implementation Method 4
bisulfite treatment occurs. Then PCR amplification is carried out to convert all the uracils to the thymines
Implementation Method 5
unmethylated cytosines in single-stranded DNA are deaminated by bisulfite under appropriate reaction conditions to give uracils
Implementation Method 6
PCR amplification is carried out to convert all the uracils to the thymines
Data Source
AI summary
The present invention provides a high-throughput sequencing method for methylated DNA, and use thereof. Particularly, the present invention provides a high-throughput sequencing method for methylated DNA, which combines methylated DNA immunoprecipitation, removal of repetitive sequences, and bisulfite treatment. The site of sequencing library will be decreased, and the cost will be reduced by using the method disclosed in the present invention.