Spatial DNA Methylation Mapping with Barcoded Capture Probes
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Solution Overview
Problem
Existing methods for analyzing DNA methylation status in biological samples are cumbersome and challenging due to the variability of methylation status among cells and the inability to distinguish methylated-cytosine bases from unmethylated-cytosine bases in standard DNA sequencing technologies, especially in tissues where spatial information is lacking.
Innovation Solution
A method involving spatial analysis using capture probes with spatial barcodes and methylated cytosines, combined with deamination and ligation techniques, to identify the methylation status of analytes by generating and extending ligation products for sequence determination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If standard DNA sequencing technologies are used, then DNA methylation analysis can be performed, but methylated-cytosine bases cannot be distinguished from unmethylated-cytosine bases
Solution Approach 1:
The patent applies preliminary action by performing deamination treatment on the analyte before sequencing. This pre-treatment converts unmethylated cytosines to uracils (which are read as thymines during sequencing), while methylated cytosines remain unchanged. This preliminary chemical modification enables the distinction between methylated and unmethylated cytosine bases that standard sequencing cannot provide, directly resolving the information loss problem.
2Measurement precision
If bisulfite conversion is used to study DNA methylation, then methylation status can be identified, but the chemical fragments DNA and is incompatible with downstream enzymes and reagents
Solution Approach 1:
The patent changes the chemical parameter of the deamination reaction by using alternative deaminating agents or conditions that are less harsh than bisulfite treatment. This parameter change allows for methylation status identification while preserving DNA integrity and maintaining compatibility with downstream enzymes and reagents, thus resolving the contradiction between measurement precision and reliability.
3Loss of information
If spatial analysis methods are used to provide location data, then spatial heterogeneity can be studied, but information on cell position in parent biological sample is lost
Solution Approach 1:
The patent implements the nested doll principle by incorporating spatial barcodes within the sequencing library preparation process. The spatial barcodes are nested within the DNA fragments during library construction, allowing both spatial location information and single-cell analyte data to be preserved and read simultaneously through sequencing. This nested approach resolves the contradiction by embedding multiple layers of information within the same molecular structure.
4Reliability
If DNA methylation analysis is performed in pathological settings, then diagnostic and therapeutic implications can be identified, but the variability of methylation status among cells makes analysis challenging
Solution Approach 1:
The patent applies segmentation by processing and analyzing individual DNA molecules or single cells separately rather than as bulk tissue. This segmentation approach allows the capture and analysis of cell-to-cell variability in methylation status, enabling the identification of rare pathological events while maintaining diagnostic and therapeutic value. The complexity is managed through automated single-cell processing workflows.
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
Provides high-resolution spatial analysis of DNA methylation status, enabling accurate identification of methylation changes in biological samples, particularly useful in cancer research and therapeutic delivery, by correlating location with gene expression and methylation status.
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
Implementation Method 4
extending the capture probe using the ligation product as a template; thereby generating an extended capture probe
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.