TAPS Method for Bisulfite-Free Cytosine Modification Detection
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Solution Overview
Problem
Current methods for DNA and RNA methylation and hydroxymethylation analysis, such as bisulfite sequencing, are harsh and degrade samples, limiting their application to low-input samples and resulting in poor sequencing quality due to incomplete conversion and high unmodified cytosine conversion rates, leading to increased costs and false detection issues.
Innovation Solution
The development of TET Assisted Pyridine borane Sequencing (TAPS) method, which uses mild reactions to detect 5-methylcytosine and 5-hydroxymethylcytosine by combining TET oxidation and reduction with borane derivatives, allowing for base-resolution detection without affecting unmodified cytosines, and variations that identify 5-formylcytosine and 5-carboxylcytosine without oxidation steps.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If bisulfite sequencing is used to detect cytosine modifications, then base-resolution detection of 5mC and 5hmC is achieved, but sample degradation exceeds 90% and sequencing quality deteriorates
Solution Approach 1:
The patent changes the chemical parameters of the detection method by using TET enzyme oxidation followed by borane reduction instead of harsh bisulfite treatment. This parameter change allows detection of 5mC and 5hmC at base resolution while maintaining sample integrity, as the enzymatic reactions occur under mild conditions that do not degrade DNA
Solution Approach 2:
The patent replaces the harsh chemical mechanism of bisulfite sequencing with a two-step enzymatic/chemical mechanism: TET enzyme oxidation of 5mC/5hmC to 5caC/5fC, followed by borane reduction to dihydrouracil. This substitution eliminates the need for extreme pH and heat conditions, preserving sample integrity while achieving the same detection goal
2Measurement precision
If complete conversion of unmodified cytosine to thymine is pursued, then detection sensitivity for 5mC and 5hmC is improved, but sequence complexity is reduced and mapping rates decrease
Solution Approach 1:
The patent extracts only the modified cytosines (5mC and 5hmC) for detection by selectively oxidizing them to 5caC/5fC and then reducing to dihydrouracil, which is read as thymine during sequencing. Unmodified cytosines remain unchanged and maintain sequence complexity, allowing both high detection sensitivity and good mapping rates
Solution Approach 2:
The patent introduces dihydrouracil as an intermediary product that serves as a marker for modified cytosines. This intermediary allows differentiation between modified and unmodified cytosines without converting all cytosines to thymine, thus preserving sequence complexity while maintaining detection sensitivity
3Measurement precision
If bisulfite treatment is applied to detect cytosine modifications, then modification detection capability is enhanced, but false detection rates increase due to incomplete conversion
Solution Approach 1:
The patent replaces the single-step bisulfite conversion mechanism with a two-step mechanism involving TET enzyme oxidation followed by borane reduction. This substitution eliminates incomplete conversion issues because the enzymatic oxidation is highly specific and efficient, and the subsequent reduction step completes the conversion to dihydrouracil, minimizing false detections
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
TAPS provides higher mapping rates, more even genome coverage, and lower sequencing costs, enabling higher quality and comprehensive methylome analyses while preserving RNA and DNA, and is compatible with low-input samples like circulating cell-free DNA and single-cell sequencing.
Implementation Method 1
converting the 5mC and 5hmC in the nucleic acid sample to 5caC and/or 5fC
Implementation Method 2
converting the 5caC and/or 5fC to DHU to provide a modified nucleic acid sample
Data Source
AI summary
This disclosure provides methods for bisulfite-free identification in a nucleic acid sequence of the locations of 5-methylcytosine, 5-hydroxymethylcytosine, 5-carboxylcytosine and 5-formylcytosine.