Direct Methylation Sequencing via Polymerase Kinetics

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Solution Overview

Problem

Current methylation profiling technologies, such as bisulfite sequencing and MeDIP, face limitations in resolution, sample requirements, and alignment issues, particularly in repetitive genomic regions, making comprehensive analysis of the human methylome challenging.

Innovation Solution

A direct methylation sequencing technology that monitors the kinetics of single polymerase molecules in real-time, using single-molecule real-time analysis to detect modifications in nucleic acid sequences, including methylated bases, with high multiplex capability.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If bisulfite sequencing is used for methylation profiling, then methylation status can be determined, but sample preparation time increases and DNA degradation occurs

Engineering Contradiction:

Improvemethylation status determinationVSAvoidsample preparation time

Core Design Contradiction:

Measurement precisionVSLoss of time

Solution Approach 1:

The patent extracts and removes the problematic bisulfite conversion step from the methylation profiling workflow. Instead of using bisulfite sequencing, the invention employs direct sequencing methods that maintain native DNA modifications, thereby eliminating the time-consuming and DNA-degrading bisulfite treatment while still enabling accurate detection of methylation status through specialized sequencing chemistry

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent changes the chemical parameters of the sequencing process by using alternative chemistry that does not require bisulfite conversion. This includes using enzymes or chemical treatments that preserve DNA integrity while still allowing differentiation between methylated and unmethylated cytosines, thus reducing preparation time and preventing DNA degradation

Inventive Principle:

Principle #35Parameter changes

2Measurement precision

If bisulfite sequencing is used for methylation profiling, then methylation status can be determined, but DNA degradation occurs necessitating large starting amounts

Engineering Contradiction:

Improvemethylation status determinationVSAvoidstarting DNA amount

Core Design Contradiction:

Measurement precisionVSQuantity of substance

Solution Approach 1:

The patent removes the DNA-degrading bisulfite conversion step from the protocol. By using direct sequencing methods that preserve DNA integrity, the invention enables accurate methylation status determination with minimal starting material, as the DNA does not undergo harsh chemical treatment that causes fragmentation and degradation

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent employs disposable, non-destructive sequencing chemistry that does not require large amounts of precious genomic DNA. The method uses enzyme-based or gentle chemical approaches that consume minimal DNA while providing sufficient signal for accurate methylation profiling, effectively replacing the need for large starting amounts required by bisulfite sequencing

Inventive Principle:

Principle #27Cheap short-living objects (Disposable)

3Productivity

If current sequencing technologies are used, then sequencing can be performed, but alignment issues occur in repetitive genomic regions

Engineering Contradiction:

Improvesequencing capabilityVSAvoidalignment accuracy in repetitive regions

Core Design Contradiction:

ProductivityVSMeasurement precision

Solution Approach 1:

The patent replaces traditional alignment-based analysis with a method that detects methylation status directly from sequencing reads without requiring precise alignment to reference genomes. This is achieved through enzymatic or chemical methods that preserve native modifications and allow direct calling of methylation status from the sequence data, bypassing the alignment step that causes problems in repetitive regions

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The patent changes the analytical parameter from alignment-dependent to alignment-independent methylation detection. By using methods that preserve DNA modifications and enable direct detection of methylated bases during sequencing, the invention achieves accurate methylation profiling in repetitive genomic regions without relying on precise alignment, thus resolving the contradiction between sequencing productivity and alignment accuracy

Inventive Principle:

Principle #35Parameter changes

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

Enables fast and economical analysis of methylation patterns, even in repetitive genomic regions, providing high-resolution detection of modifications in nucleic acids.

Implementation Method 1

Methodology Applied

Scientific EffectSingle-molecule real-time analysis:

Data Source

PatentUS12527665B2Methods for identifying modified bases in nucleic acid templates

Publication Date: 2026.01.20 PACIFIC BIOSCIENCES OF CALIFORNIA INC

AI summary

Methods, compositions, and systems are provided for characterization of modified nucleic acids. In certain preferred embodiments, single molecule sequencing methods are provided for identification of modified nucleotides within nucleic acid sequences. Modifications detectable by the methods provided herein include chemically modified bases, enzymatically modified bases, abasic sites, non-natural bases, secondary structures, and agents bound to a template nucleic acid.