TAPS cfDNA Sequencing for Methylation Detection
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Solution Overview
Problem
Current methods for detecting early-stage cancer, particularly hepatocellular carcinoma (HCC) and pancreatic ductal adenocarcinoma (PDAC), are limited by low sensitivity and specificity, and existing cfDNA methylation sequencing techniques are low-depth, targeted, or qualitative, failing to accurately capture the cfDNA methylome due to DNA damage and high costs.
Innovation Solution
The development of TET-assisted Pyridine Borane Sequencing (TAPS) for cfDNA, which isolates and sequences cell-free DNA to obtain high-quality, whole-genome methylation signatures, enabling the identification of methylation biomarkers, tissue of origin, and DNA fragmentation profiles for accurate cancer detection and classification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If bisulfite sequencing is used to determine DNA methylation, then methylation detection capability is improved, but DNA damage increases and cost increases
Solution Approach 1:
The patent extracts and removes the harmful bisulfite treatment step from the methylation sequencing process. Instead of using bisulfite conversion, the invention employs TAPS (TET-assisted pyridine borane sequencing) which achieves methylation detection without the DNA-damaging chemical treatment, thereby eliminating the harmful factor while preserving the detection capability
Solution Approach 2:
The patent introduces TET enzymes and pyridine borane reagents as intermediary substances that mediate the methylation detection process. These intermediaries enable the conversion and detection of methylated cytosines through enzymatic reactions and selective chemical modifications, providing an alternative pathway that avoids direct DNA damage from bisulfite while maintaining measurement precision
2Measurement precision
If bisulfite sequencing is used to determine DNA methylation, then methylation detection capability is improved, but sequencing cost increases
Solution Approach 1:
The patent employs disposable, cost-effective reagents including pyridine borane and TET enzymes that can be used in a single sequencing run. These reagents provide the necessary methylation detection capability without the high costs associated with bisulfite sequencing, enabling economical whole-genome methylation analysis
Solution Approach 2:
The patent changes the chemical parameters of the sequencing process by replacing bisulfite treatment with enzymatic oxidation followed by pyridine borane modification. This parameter change transforms the detection chemistry to be more cost-effective while maintaining the ability to detect methylation patterns across the whole genome with base-resolution precision
3Loss of energy
If low-depth or targeted sequencing is used for cfDNA methylation, then sequencing cost is reduced, but measurement precision deteriorates
Solution Approach 1:
The patent creates a universal sequencing method (TAPS) that can perform whole-genome methylation sequencing at base-resolution without requiring targeted enrichment. This multi-functional approach allows the same protocol to achieve comprehensive genomic coverage with high precision, eliminating the need to choose between cost-effective low-depth sequencing and precise targeted sequencing
4Loss of energy
If qualitative enrichment-based sequencing is used for cfDNA methylation, then sequencing cost is reduced, but measurement precision deteriorates
Solution Approach 1:
The patent extracts and removes the qualitative enrichment step from the sequencing workflow. By using TAPS, the method directly sequences methylated bases across the entire genome without requiring prior enrichment or selection, thereby achieving both cost-effectiveness and base-resolution quantitative precision simultaneously
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 achieves high unique mapping rates and depth, providing comprehensive cfDNA methylation analysis, enabling the detection of early-stage cancers with high accuracy and reducing sequencing costs, while maintaining genetic information integrity.
Implementation Method 1
TET-assisted Pyridine Borane Sequencing (TAPS)... converting 5mC and/or 5hmC residues in the sample to DHU residues
Implementation Method 2
TET-assisted Pyridine Borane Sequencing (TAPS)... converting 5mC and/or 5hmC residues in the sample to DHU residues
Data Source
AI summary
The present disclosure provides compositions and methods related to TET-assisted Pyridine Borane Sequencing (TAPS). In particular, the present disclosure provides optimized TAPS for cfDNA (cfTAPS), which provides high-quality and high-depth whole-genome cell-free methylomes. The compositions and methods provided herein facilitate the acquisition of multimodal information about cfDNA characteristics, including DNA methylation, tissue of origin, and DNA fragmentation for the diagnosis and treatment of disease.