Six-Nucleobase DNA Sequencing for Direct Methylation and SNP Detection
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Solution Overview
Problem
Current methods for detecting 5-methylcytosine nucleobases, such as whole-genome bisulfite sequencing (WGBS), suffer from issues like overestimation of 5-methylcytosine abundance, GC bias, and dropout of nonmethylated regions, making simultaneous detection of SNPs and methylation challenging, especially in small sample amounts.
Innovation Solution
A method involving the conversion of modified nucleobases to unnatural nucleobases using selective chemical reagents, forming a novel 'six-nucleobase' alphabet for sequencing-by-synthesis (SBS) that allows direct detection of 5-methylcytosine without losing SNP information.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If whole-genome bisulfite sequencing (WGBS) is used to detect 5-methylcytosine, then methylation detection is achieved, but SNP information is lost and 5-methylcytosine abundance is overestimated
Solution Approach 1:
The patent divides the detection process into separate functional modules: (1) a detection module that identifies 5-methylcytosine through selective chemical conversion to orthogonal nucleobases, and (2) a sequencing module that preserves SNP information by using a six-nucleobase alphabet. This segmentation allows each module to perform its specific function without interfering with the other, thereby achieving both accurate methylation detection and SNP preservation simultaneously.
Solution Approach 2:
The patent introduces orthogonal nucleobases (X and Y) as intermediary elements that mediate between the detection of 5-methylcytosine and the preservation of SNP information. These orthogonal nucleobases serve as temporary markers during the detection process, allowing 5-methylcytosine to be identified without permanently altering the original genomic information. The orthogonal nucleobases can be selectively incorporated and later removed or converted, thus acting as reversible intermediaries that enable dual detection capabilities.
2Measurement precision
If bisulfite treatment is applied to convert cytosine to uracil, then 5-methylcytosine positions are marked, but cytosine-to-thymine SNPs are masked
Solution Approach 1:
The patent changes the chemical parameter of nucleobase conversion by replacing the traditional bisulfite-induced cytosine-to-uracil conversion with a selective chemical conversion system that transforms 5-methylcytosine into orthogonal nucleobases (X and Y). This parameter change in the conversion mechanism allows for differential marking: 5-methylcytosine is converted to orthogonal bases while unmodified cytosine remains unchanged or is converted to different markers, thereby preserving the ability to distinguish between methylated positions and cytosine-to-thymine SNPs.
Solution Approach 2:
Instead of converting all cytosines to uracils and then inferring methylation status indirectly (inverse detection), the patent inverts the approach by directly converting 5-methylcytosine to orthogonal nucleobases that can be directly read during sequencing. This inversion from indirect inference to direct detection eliminates the masking effect on SNP information, as the conversion is specific to methylated cytosines and does not uniformly alter all cytosine positions.
3Measurement precision
If parallel whole-genome sequencing library is prepared for SNP detection, then SNP detection is enabled, but sample amount requirements increase
Solution Approach 1:
The patent creates a universal sequencing system that performs multiple functions simultaneously: it can detect 5-methylcytosine methylation, identify cytosine-to-thymine SNPs, and generate genome sequence information all in a single sequencing run. The six-nucleobase alphabet and orthogonal nucleobase system are designed to be universally applicable to all these detection tasks, eliminating the need for separate parallel libraries and reducing sample amount requirements by consolidating multiple detection goals into one multi-functional assay.
Solution Approach 2:
The patent merges the methylation detection function and SNP detection function into a single integrated sequencing process. By incorporating orthogonal nucleobase markers that are compatible with standard sequencing chemistries, the system combines what were previously separate detection pipelines (WGBS for methylation and WGS for SNPs) into one unified approach, thereby reducing the total sample input needed and simplifying the experimental workflow.
4Ease of operation
If conventional four-nucleobase sequencing is used, then sequencing simplicity is maintained, but 5-methylcytosine detection capability is lost
Solution Approach 1:
The patent creates a composite nucleic acid system that combines the familiar four natural nucleobases (A, T, C, G) with two additional orthogonal nucleobases (X, Y). This composite six-nucleobase system maintains compatibility with standard DNA polymerases and sequencing chemistries while adding the functional capability to detect 5-methylcytosine. The orthogonal nucleobases are designed to base-pair with each other specifically, creating a composite structure that can be incorporated into standard sequencing workflows without requiring complete overhaul of the sequencing infrastructure.
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 provides an information-rich genome sequence that can detect 5-methylcytosine and SNPs simultaneously, overcoming the limitations of inverse detection methods, and is suitable for small sample types like cell-free DNA and single-cell samples.
Implementation Method 1
removing the modified nucleobase is accomplished by a glycosylase selected from the group consisting of ROS1 DNA glycosylase, DME DNA glycosylase, DML2 DNA glycosylase, and DML3 DNA glycosylase
Implementation Method 2
converting the paired nucleobase into an orthogonal nucleobase... converting the paired nucleobase is accomplished with chemical reagents
Data Source
AI summary
Embodiments of the present disclosure relate to six-nucleobase libraries having a third Watson-Crick base pair. Also provided herein are methods to prepare such six-nucleobase libraries, and their use for sequencing and modified nucleobase detection applications.