Spatial DNA Methylation Mapping With Enzyme-Compatible Deamination
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Solution Overview
Problem
Existing methods fail to provide spatial information on DNA methylation status within a tissue sample, complicating the analysis of methylation in heterogeneous biological samples, particularly in pathological settings like cancer, due to the challenges of distinguishing methylated and unmethylated cytosines and the harsh conditions of bisulfite treatment.
Innovation Solution
A method involving spatial analysis with capture probes containing spatial barcodes and capture domains, combined with deamination and ligation techniques, to identify the methylation status of analytes in a biological sample, allowing for the determination of methylation patterns at specific locations within the sample.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If bisulfite treatment is used to distinguish methylated cytosines, then methylation status can be identified, but DNA is fragmented and harsh conditions are created that are incompatible with downstream enzymes
Solution Approach 1:
The method performs preliminary deamination of cytosines to uracils before any enzymatic steps. This preliminary action converts the problematic methylated/unmethylated cytosine distinction into a methylated cytosine vs. uracil distinction, allowing downstream enzymes to work on intact DNA without bisulfite treatment
Solution Approach 2:
The invention introduces an intermediary chemical step (deamination) that transforms the DNA structure in a controlled manner. This intermediary action creates a state where methylation status can be detected through subsequent enzymatic reactions without requiring harsh bisulfite treatment, thus mediating between the need for methylation detection and enzyme compatibility
2Loss of information
If spatial analysis methods are used to provide location data, then spatial information is obtained, but the number of analytes that can be analyzed is limited
Solution Approach 1:
The method segments the analysis process into distinct modular steps: spatial barcode assignment, deamination, probe hybridization, and sequencing. This segmentation allows different analytes to be processed through the same spatial framework, increasing versatility while maintaining spatial information
Solution Approach 2:
The spatial barcode system serves as a universal platform that can track multiple different analytes simultaneously. The same spatial barcoding infrastructure is used regardless of which analyte is being studied, making the system adaptable to various methylation analysis needs while preserving location data
3Quantity of substance
If single-cell analysis is performed to obtain detailed analyte data, then comprehensive analyte information is obtained, but spatial position information is lost
Solution Approach 1:
The invention merges single-cell analytical power with spatial context by combining spatial barcoding with comprehensive analyte detection. Each cell's analyte profile is captured and linked to its spatial position through the barcode system, merging the advantages of both approaches into a unified method
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
Enables accurate and cost-effective identification of DNA methylation status with spatial resolution, facilitating analysis in normal physiological conditions and pathophysiological settings, including cancer, by correlating gene expression and methylation status with image location.
Implementation Method 1
hybridizing the ligation product to the capture probe
Implementation Method 2
deaminating the analyte in the biological sample
Implementation Method 3
ligating the first probe and the second probe, thereby generating a ligation product
Data Source
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AI summary
Provided herein are methods of identifying a methylation status of an analyte in a biological sample. Also provided herein are methods that combine identifying the methylation status with spatial technology to identify the location of a methylation status in a biological sample.