Nucleic Acid Methylation Profiling With 5hmC Partitioning
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Solution Overview
Problem
Existing nucleic acid methylation detection methods, such as bisulfite and EM-Seq, struggle to resolve the methylation status of unmethylated cytosine (C), 5-methylcytosine (5mC), and 5-hydroxymethylcytosine (5hmC) at single nucleotide resolution, leading to low sensitivity and high assay costs due to sample splitting and incomplete molecular information.
Innovation Solution
A method integrating methylation-specific base conversion with 5hmC-based partitioning into a single workflow, allowing for the resolution of unmethylated C, 5mC, and 5hmC bases on individual sequence reads by partitioning nucleic acids based on 5hmC presence, converting 5mC or unmethylated C base pairing specificity, amplifying, and sequencing to determine methylation status.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If sample splitting is used to detect different methylation statuses, then detection coverage is improved, but sensitivity decreases and assay costs increase
Solution Approach 1:
The patent divides the detection process into two separate assays: a first assay that detects unmethylated cytosines (C) by converting them to uracil, and a second assay that detects methylated cytosines (5mC) by converting them to a different state. This segmentation allows each assay to be optimized for its specific target, improving sensitivity for each methylation status while maintaining comprehensive detection coverage through the combination of both assays.
2Measurement precision
If multiple conversion methods are used to resolve C/5mC/5hmC status, then measurement precision is improved, but device complexity increases
Solution Approach 1:
The patent segments the complex task of detecting three methylation statuses (C, 5mC, 5hmC) into two simpler, specialized assays. The first assay uses a conversion method specific for unmethylated cytosines, while the second assay uses a different conversion method for methylated cytosines. This segmentation reduces the complexity of individual assays while achieving comprehensive resolution of all methylation statuses through their combination.
3Measurement precision
If subtractive sequencing workflows are used, then measurement precision is improved, but loss of substance increases
Solution Approach 1:
Instead of using a subtractive workflow that requires splitting and processing multiple subsamples (leading to sample loss), the patent employs two parallel conversion-based assays that can be performed on separate aliquots of the original sample. Each assay preserves molecular information by converting specific methylation statuses to detectable states, and the results are integrated to provide complete methylation pattern information without the cumulative sample loss associated with sequential subtractive processing.
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 simultaneous detection of C, 5mC, and 5hmC methylation patterns on an individual molecule level, improving sensitivity and reducing costs by integrating partitioning and conversion steps into a unified process.
Implementation Method 1
subjecting the nucleic acids to a conversion procedure that selectively converts the base pairing specificity of 5-methylcytosines (5mC) or unmethylated cytosines (C) in the nucleic acids
Data Source
AI summary
The disclosure relates to methods for determining the methylation profile of nucleic acids. The methods use base conversion methods in combination with methylation-based partitioning methods to resolve multiple types of methylation in a single workflow.