DHU-Tolerant TAPS Sequencing for Low-Input Methylation Analysis
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Solution Overview
Problem
Current bisulfite sequencing methods, such as bisulfite sequencing, TET-assisted bisulfite sequencing, and oxidative bisulfite sequencing, face challenges including DNA degradation, poor sequencing quality, and false detection due to incomplete conversion of unmodified cytosine to thymine, particularly in low-input samples like clinical samples and single-cell sequencing, leading to underrepresentation of highly methylated regions.
Innovation Solution
The method introduces dihydrouracil (DHU) residues into nucleic acid samples using TET-assisted Pyridine Borane Sequencing (TAPS) and variants like TAPSβ, employing polymerases tolerant of DHU residues for synthesis and exponential amplification, followed by sequencing with borane reducing agents to convert 5-carboxylcytosine and 5-formylcytosine to DHU.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If bisulfite treatment is used to convert unmethylated cytosine to thymine, then methylation analysis capability is improved, but DNA degradation exceeds 90% under acidic and thermal conditions
Solution Approach 1:
The patent introduces an intermediary chemical reaction system using TET enzymes and borane reagents that mediates the conversion of methylated cytosines to detectable forms without requiring harsh bisulfite treatment. This intermediary pathway allows specific detection of 5mC and 5hmC while preserving overall DNA integrity.
Solution Approach 2:
The invention changes the chemical parameters of the detection system by replacing acidic bisulfite treatment with enzymatic oxidation followed by borane reduction. This parameter change operates under milder conditions (neutral pH, lower temperature) that preserve DNA while maintaining detection capability.
2Measurement precision
If complete conversion of unmodified cytosine to thymine is pursued, then methylation detection accuracy is improved, but sequence complexity reduces leading to poor sequencing quality and low mapping rates
Solution Approach 1:
The patent extracts only the methylated cytosine detection function from the complete cytosine-to-thymine conversion process. By using TET enzymes to specifically oxidize 5mC and 5hmC followed by borane reduction, the method isolates the detection of modified cytosines while leaving unmodified cytosines intact, thereby maintaining sequence complexity and sequencing quality.
Solution Approach 2:
The detection process is segmented into specific enzymatic steps: TET1/2/3 oxidize only methylated cytosines to 5fC/5caC, then borane reagents reduce these to DHU. This segmentation allows selective modification of target bases without affecting the entire cytosine population, preserving genome-wide sequence information.
3Quantity of substance
If bisulfite sequencing is used for whole genome methylation analysis, then comprehensive coverage is improved, but sequencing cost increases and ability to call variants reduces
Solution Approach 1:
The patent uses enzymatic intermediaries (TET enzymes and borane reagents) to create detectable differences between methylated and unmethylated cytosines without requiring complete base conversion. This intermediary approach maintains better sequencing library complexity and variant calling capability while reducing costs compared to comprehensive bisulfite sequencing.
4Measurement precision
If bisulfite treatment is applied to low-input samples, then methylation analysis is enabled, but DNA degradation severely limits application to clinical samples and single-cell sequencing
Solution Approach 1:
The invention changes the reaction parameters from harsh acidic conditions to mild enzymatic conditions (neutral pH, physiological temperature). This allows the method to work with low-input samples including clinical specimens and single cells, as the DNA does not undergo catastrophic degradation during 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
This approach improves sequencing coverage and quality of highly methylated regions, reducing sequencing costs and enhancing the ability to detect 5-methylcytosine and 5-hydroxymethylcytosine without degrading DNA, making it suitable for low-input samples and various sequencing methods, including NGS.
Implementation Method 1
contacting the oxidized nucleic acid sample with a borane reducing agent to convert the 5-caC and 5fC to dihydrouracil (DHU)
Implementation Method 2
5hmC is generated from 5mC by the ten-eleven translocation (TET) family dioxygenases. TET can further oxidize 5hmC to 5-formylcytosine (5fC) and 5-carboxylcytosine (5 caC)
Data Source
AI summary
Methods of amplifying libraries after introduction of dihydrouracil (DHU) residues by methods such as TET-assisted Pyridine Borane Sequencing (TAPS), and variants of TAPS including TAPS with blocking by β-glucosylation (TAPSβ) and Chemically-assisted Pyridine Borane Sequencing (CAPS) are described. The methods comprise introducing DHU residues into a nucleic acid sample and preparation of a sequencing library by a complementary strand synthesis step reaction with a first polymerase or polymerase mixture that is tolerant of DHU residues and/or products resulting from the introduction of the DHU residues and/or the TAPS process followed by exponential amplification. Improved methods for conversion of oxidized nucleotide residues to DHU are also described.