Long-Read Cytosine Modification Sequencing Without Bisulfite Damage
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Solution Overview
Problem
Current long-read sequencing methods for detecting cytosine modifications like 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are limited by high sample input requirements, computational complexity, and DNA degradation, making them unsuitable for low-input clinical samples and complicating accurate, cost-effective detection.
Innovation Solution
A bisulfite-free method, named lrTAPS, performs enzymatic conversions in a single tube to convert 5mC and 5hmC to dihydrouracil (DHU) or thymine (T) for precise detection, allowing high sensitivity and specificity without affecting unmodified cytosines, suitable for both PacBio SMRT and Oxford Nanopore sequencing platforms.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If current long-read sequencing methods are used to detect cytosine modifications, then detection capability is provided, but high sample input requirements and DNA degradation occur
Solution Approach 1:
The patent applies preliminary action by performing enzymatic conversion of cytosine modifications to uracil before sequencing. This pre-treatment step converts modified cytosines (5mC, 5hmC) into detectable uracil residues through enzymatic reactions (TET oxidation followed by APE1 cleavage and uracil-DNA glycosylase treatment), enabling sensitive detection of low-input samples without requiring high sample quantities
Solution Approach 2:
The patent uses enzymatic intermediaries (TET enzymes, APE1, uracil-DNA glycosylase) as mediators to convert cytosine modifications into detectable forms. These enzymatic intermediaries facilitate the transformation of epigenetic marks into sequence-detectable uracil residues, enabling accurate detection while preserving DNA integrity and reducing sample input requirements
2Measurement precision
If current long-read sequencing methods are used to detect cytosine modifications, then detection is provided, but computational complexity increases
Solution Approach 1:
The patent extracts the computational complexity from the sequencing analysis by performing enzymatic conversion of cytosine modifications to uracil before sequencing. This physical/chemical preprocessing step converts epigenetic information into direct sequence variations (C-to-U transitions), eliminating the need for complex computational algorithms to infer modifications from sequencing data
Solution Approach 2:
The patent replaces computational analysis with enzymatic chemistry. Instead of using complex bioinformatics pipelines to detect and analyze cytosine modifications, the method uses enzymatic reactions (TET oxidation, APE1 cleavage, uracil-DNA glycosylase treatment) to physically convert modifications into detectable uracil residues, substituting mechanical/chemical processes for computational complexity
3Measurement precision
If current sequencing methods are used, then detection is provided, but sequencing costs increase
Solution Approach 1:
The patent merges multiple steps (enzymatic conversion, DNA repair, and sequencing preparation) into a unified workflow that is compatible with standard long-read sequencing protocols. By integrating the enzymatic conversion of cytosine modifications directly into the library preparation process for PacBio or Oxford Nanopore sequencing, the method achieves comprehensive modification detection without requiring separate specialized assays, thereby reducing overall sequencing costs
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
lrTAPS enables accurate, long-read, cost-effective detection of cytosine modifications with high sensitivity and specificity, preserving RNA and DNA integrity, and facilitating comprehensive methylome analyses at lower sequencing costs.
Implementation Method 1
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 (5caC)
Implementation Method 2
A bisulfite-free method, named lrTAPS, performs enzymatic conversions in a single tube to convert 5mC and 5hmC to dihydrouracil (DHU) or thymine (T) for precise detection
Data Source
AI summary
The present disclosure provides a bisulfite-free, long-read, base-resolution method named long-read TAPS (lrTAPS) for detecting 5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in a nucleic acid sequence. lrTAPS comprises mild enzymatic and chemical reactions to detect 5mC and 5hmC, the two major epigenetic marks found in the mammalian genome, quantitatively at base-resolution without affecting unmodified cytosine.