Methylation Sequencing with Enzymatic Conversion for cfDNA Integrity

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Solution Overview

Problem

Existing methylation sequencing methods, particularly bisulfite conversion, cause significant DNA degradation in cell-free DNA (cfDNA), leading to loss of fragment length information and requiring large blood volumes or low-depth coverage, and are costly and error-prone for analyzing methylation patterns in genetically heterogeneous samples.

Innovation Solution

A minimally-destructive conversion method using enzymatic conversion (e.g., TAPS or CAPS) converts unmethylated cytosines to uracils, followed by PCR amplification, probing with nucleic acid probes, and deep sequencing (>100×) to determine methylation profiles, incorporating unique molecular identifiers and duplex sequencing for error correction.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If bisulfite conversion is used for methylation sequencing, then methylation patterns can be analyzed, but cfDNA degrades significantly (>90% loss) and fragment length information is lost

Engineering Contradiction:

Improvemethylation pattern analysis accuracyVSAvoidcfDNA integrity

Core Design Contradiction:

Measurement precisionVSLoss of substance

Solution Approach 1:

The patent changes the chemical parameters of the conversion process by using enzymatic conversion (TAPS or CAPS) instead of bisulfite conversion. This enzymatic approach converts unmethylated cytosines to uracils under milder conditions that preserve cfDNA integrity and fragment length information while still enabling methylation pattern analysis through subsequent sequencing.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent replaces the harsh chemical mechanism of bisulfite conversion with a biological enzymatic mechanism. The enzymatic conversion process uses enzymes to selectively convert unmethylated cytosines, providing a gentler alternative that maintains DNA integrity while achieving the same analytical goal of methylation detection.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

2Ease of manufacture

If bisulfite conversion is performed before library construction, then single-stranded DNA libraries can be built, but endpoint information on degraded fragments is lost

Engineering Contradiction:

Improvelibrary construction feasibilityVSAvoidfragment endpoint information

Core Design Contradiction:

Ease of manufactureVSLoss of information

Solution Approach 1:

The patent performs the conversion after library construction rather than before, reversing the conventional sequence. By constructing the library first with intact cfDNA (preserving endpoint information) and then performing enzymatic conversion on the library, the method maintains fragment length information while enabling methylation analysis.

Inventive Principle:

Principle #10Preliminary action

3Measurement precision

If large blood volumes are collected to achieve high-depth coverage, then sequencing accuracy improves, but sample collection becomes more invasive and costly

Engineering Contradiction:

Improvesequencing accuracyVSAvoidblood volume required

Core Design Contradiction:

Measurement precisionVSQuantity of substance

Solution Approach 1:

The patent converts the limitation of low cfDNA quantity in plasma into an advantage by using enzymatic conversion that preserves fragment integrity. This allows high-depth sequencing (>100×) to be achieved on small blood volumes because the method maximizes information extraction from each cfDNA molecule without requiring large sample inputs.

Inventive Principle:

Principle #22Blessing in disguise (Convert harm into benefit)

4Reliability

If duplex sequencing with unique molecular identifiers is implemented, then sequencing errors are reduced, but protocol complexity increases

Engineering Contradiction:

Improvesequencing accuracyVSAvoidprotocol complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The patent merges duplex sequencing with enzymatic methylation conversion into a unified protocol. By integrating the UMI tagging, enzymatic conversion, and methylation analysis steps into a coordinated workflow, the method achieves high accuracy through error correction while managing protocol complexity through systematic integration of steps.

Inventive Principle:

Principle #5Merging (Combining)

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

Preserves cfDNA integrity, improves sequencing accuracy, reduces sample requirements, and enables high-depth, accurate methylation profiling, facilitating early cancer detection and monitoring through improved methylation state analysis.

Implementation Method 1

converting unmethylated cytosines to uracils in the nucleic acid molecule using a minimally-destructive conversion method

Methodology Applied

Scientific EffectEnzymatic conversion: Enzyme

Data Source

PatentUS20260085351A1Methods and systems for high-depth sequencing of methylated nucleic acid

Publication Date: 2026.03.26 FREENOME HOLDINGS INC

AI summary

Methods and systems provided herein address current limitations of bisulfite-based methylation sequencing by improving the quality and accuracy of nucleic acid methylation sequencing and uses thereof for detection of disease. Methods that include minimally-destructive conversion methods for methylation sequencing as well as specialized UMI adapters provide for improved quality of sequencing libraries and sequencing information. Greater accuracy and more complete methylation-state information permits higher quality feature generation for use in machine learning models and classifier generation.