Targeted Nucleic Acid Sequencing for Accurate Base Modification Detection

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Solution Overview

Problem

Current array-based methods for identifying base modifications in a genome, such as methylated cytosines, are expensive, have limited accuracy and dynamic range, and cannot identify other base modifications or determine the type of methylation present, while reduced representation bisulfite sequencing (RRBS) is technically limited due to enrichment difficulties and requires large amounts of sequencing.

Innovation Solution

A method involving the ligation of adapters to target polynucleotides, followed by the creation of complementary adapter-target polynucleotides with protective groups, and sequencing the target polynucleotide without sequencing the complementary strand, allowing for targeted and accurate assessment of base modifications.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Ease of operation

If array-based methods are used for identifying base modifications, then the process is simple, but accuracy and dynamic range are limited

Engineering Contradiction:

Improvesimplicity of methodVSAvoidaccuracy of base modification detection

Core Design Contradiction:

Ease of operationVSMeasurement precision

Solution Approach 1:

The patent introduces an intermediary enzymatic conversion step (using enzymes such as methyltransferases or glycosylases) between the target DNA and the detection system. This intermediary step enables accurate detection of various base modifications by converting them into detectable forms without requiring complex array-based approaches, thereby improving measurement precision while maintaining operational simplicity.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Productivity

If RRBS is used for targeted sequencing, then sequencing focus is improved, but enrichment consistency and accuracy deteriorate

Engineering Contradiction:

Improvetargeted sequencing efficiencyVSAvoidenrichment accuracy at regions of interest

Core Design Contradiction:

ProductivityVSMeasurement precision

Solution Approach 1:

The patent changes the biochemical parameters of the enrichment process by using enzymatic conversion under controlled conditions rather than bisulfite treatment. This allows for consistent and accurate enrichment of regions of interest while maintaining the targeted sequencing efficiency of RRBS, resolving the contradiction between productivity and measurement precision.

Inventive Principle:

Principle #35Parameter changes

3Adaptability or versatility

If RRMS is used for whole genome sequencing, then comprehensive base modification detection is achieved, but sequencing cost and complexity increase

Engineering Contradiction:

Improvecomprehensive detection capabilityVSAvoidsequencing requirement complexity

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The patent extracts and sequences only the relevant portions of the genome containing base modifications after enzymatic conversion, rather than requiring whole genome sequencing. This extraction approach enables comprehensive detection of base modifications while significantly reducing sequencing complexity and cost, thereby resolving the contradiction between adaptability and device complexity.

Inventive Principle:

Principle #2Taking out (Extraction)

4Quantity of substance

If amplification and bisulfite conversion are used in sequencing, then DNA quantity is increased, but assay variance increases

Engineering Contradiction:

ImproveDNA quantity for sequencingVSAvoidassay consistency

Core Design Contradiction:

Quantity of substanceVSReliability

Solution Approach 1:

The patent uses a disposable enzymatic conversion step that directly prepares the DNA for sequencing without requiring amplification or bisulfite conversion. This approach maintains DNA integrity and reduces assay variance while still providing sufficient material for sequencing, thereby improving reliability without sacrificing DNA quantity.

Inventive Principle:

Principle #27Cheap short-living objects (Disposable)

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 cost-effective and accurate sequencing and detection of base modifications in specific genomic regions, avoiding amplification and bisulfite conversion-related variance.

Implementation Method 1

contacting the target polynucleotide with one or more adapters in the presence of a first ligase under conditions promoting ligation of the one or more adapters to the target polynucleotide

Methodology Applied

Scientific EffectLigation: Enzyme

Implementation Method 2

contacting the adapter-target polynucleotide with a first polymerase under conditions promoting creation of a complementary adapter-target polynucleotide

Methodology Applied

Scientific EffectPolymerization: Enzyme

Data Source

PatentUS20260071273A1Methods for nucleic acid sequencing

Publication Date: 2026.03.12 NANOHYB LLC

AI summary

Provided herein, inter alia, are methods for sequencing a nucleic acid (e.g., a target polynucleotide). In addition, provided herein are methods for detecting a base modification in polynucleotide. The methods provided herein are useful for the efficient sequencing of biological samples.