CpG Methylation-Enriched DNA Library Construction for Sequencing Cost Reduction
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods are inadequate for efficiently identifying and distinguishing between differentially methylated genomic regions across samples, which is crucial for various applications including disease diagnosis and sequencing cost reduction.
Innovation Solution
The use of CpG methylation-sensitive restriction enzymes, such as AluI, DdeI, HpyCH4IV, HpaII, HaeIII, RsaI, and Sau3AI, to generate cut or nick sites in DNA, followed by adapter ligation and sequencing, allows for the identification of differentially methylated regions by comparing sequencing reads to a reference genome.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If current methods are used to identify differentially methylated regions, then comprehensive genomic coverage is achieved, but sequencing costs increase and depth of coverage decreases
Solution Approach 1:
The method extracts and enriches specifically for differentially methylated regions by using methylation-sensitive restriction enzymes to cut only at unmethylated sites, followed by adapter ligation and selective amplification. This extraction approach isolates the target regions of interest from the entire genome, enabling focused sequencing that reduces costs while maintaining identification accuracy.
Solution Approach 2:
The method applies local quality enhancement by treating different genomic regions differently based on their methylation status. Methylation-sensitive restriction enzymes selectively cut unmethylated regions, while methylated regions remain intact. This creates locally differentiated DNA fragments that can be selectively processed and sequenced, improving sequencing efficiency for regions of interest.
2Loss of information
If whole genome sequencing is performed to identify methylation patterns, then complete genomic information is obtained, but sequencing depth and cost-effectiveness deteriorate
Solution Approach 1:
The method extracts only the biologically relevant information regarding differential methylation by using methylation-sensitive restriction enzymes to selectively cut DNA at unmethylated sites. This extraction approach obtains complete information about methylation status in the regions of interest without requiring sequencing of the entire genome, thereby reducing resource consumption while maintaining information completeness for the targeted analysis.
Solution Approach 2:
The method segments the genome into methylated and unmethylated regions based on restriction enzyme cutting patterns. By dividing the genomic information into these distinct segments and selectively processing the cut fragments through adapter ligation and amplification, the method obtains complete methylation pattern information while sequencing only a fraction of the total genome, reducing resource requirements.
3Reliability
If methylation status is not used for enrichment, then all DNA molecules are sequenced, but sequencing costs increase and depth of coverage decreases
Solution Approach 1:
The method uses local quality differentiation through methylation-sensitive restriction enzymes that recognize and cut only unmethylated DNA sequences. This creates locally distinct DNA fragments with different properties (cut vs. uncut) that can be selectively enriched. The methylation status information is thus converted into physical differences in DNA fragments, enabling reliable determination of methylation patterns while improving sequencing throughput and cost efficiency through targeted enrichment.
Solution Approach 2:
The method introduces methylation-sensitive restriction enzymes as intermediaries that mediate between the methylation status of DNA and the sequencing process. These enzymes act as biological mediators that convert epigenetic information into physical DNA fragment differences, which can then be selectively amplified and sequenced. This intermediary approach maintains accurate methylation status determination while enabling cost-effective, high-throughput sequencing of enriched targets.
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 effectively enriches or depletes CpG-methylated DNA, enabling the identification of genomic regions with differential methylation patterns, thereby aiding in disease diagnosis and reducing sequencing costs by distinguishing between DNA from different cell types or organisms.
Implementation Method 1
contacting the samples with one or more CpG methylation-sensitive restriction enzyme to generate cut sites or nick sites in the DNA at enzyme recognition sites
Implementation Method 2
ligating adapters to the cut sites or nick sites to generate DNA libraries
Data Source
AI summary
The present invention provides methods for identifying genomic regions that are differentially CpG methylated in two samples. Also provided are novel methods for generating a DNA library that is enriched for or depleted of CpG-methylated DNA and enzyme compositions for use in the disclosed methods.