Double-Stranded Nucleic Acid Molecule for Removing Free Adapters in DNA Libraries

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Solution Overview

Problem

The reliability of next-generation sequencing (NGS) is compromised by free adapters that are not ligated to DNA inserts, leading to issues like index hopping and unique molecular identifier (UMI) mix, which reduce the accuracy of sequencing results.

Innovation Solution

A double-stranded nucleic acid molecule with a type IIs restriction enzyme recognition site and a complementary sequence, along with DNA ligase and a type IIs restriction enzyme, is used to selectively remove free adapters by forming a complex with the adapters and cutting them using the restriction enzyme.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If adapters are added to DNA fragments for NGS library construction, then sequencing throughput is improved, but free adapters cause index hopping and UMI mix reducing reliability

Engineering Contradiction:

Improvesequencing throughputVSAvoidsequencing reliability

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent extracts and removes free adapters from the DNA library using a specific methodology involving adapter-specific primers and PCR amplification. This selectively eliminates the harmful free adapters while preserving the useful adapter-DNA insert complexes, thereby maintaining high sequencing throughput while improving reliability by preventing index hopping and UMI mix artifacts.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent introduces an intermediary approach using adapter-specific primers and controlled PCR conditions as mediators to selectively amplify and remove free adapters. This intermediary mechanism allows for the specific targeting and removal of free adapters without affecting the overall library structure, thus resolving the contradiction between maintaining high throughput and ensuring reliability.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Ease of manufacture

If free adapters remain in the DNA library, then library construction is simpler, but they cause UMI mix and reduce data accuracy

Engineering Contradiction:

Improvelibrary construction simplicityVSAvoidsequencing data accuracy

Core Design Contradiction:

Ease of manufactureVSMeasurement precision

Solution Approach 1:

The patent applies preliminary action by performing adapter removal treatment before sequencing analysis. The methodology includes preliminary steps of treating the library with adapter-specific primers and controlled PCR to eliminate free adapters prior to downstream analysis. This preliminary action ensures that only properly ligated adapter-DNA complexes are sequenced, thereby improving measurement precision while maintaining ease of manufacture through a straightforward treatment protocol.

Inventive Principle:

Principle #10Preliminary action

3Productivity

If multiplexing is implemented to increase sample throughput, then sequencing efficiency is improved, but processing complexity increases due to demultiplexing requirements

Engineering Contradiction:

Improvesample throughputVSAvoidprocessing complexity

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The patent extracts and removes free adapters that would otherwise cause index hopping and UMI mix during multiplexed sequencing. By selectively eliminating these problematic free adapters through adapter-specific PCR treatment, the patent simplifies the demultiplexing process and reduces processing complexity while maintaining high sample throughput efficiency.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent employs a disposable treatment approach using adapter-specific primers and controlled PCR as a one-time preprocessing step. This simple, inexpensive treatment effectively removes free adapters and eliminates the need for complex ongoing management of multiplexed libraries, thereby reducing processing complexity while maintaining high productivity.

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

This method effectively prevents index hopping and UMI mix by specifically cutting and removing free adapters, thereby enhancing the reliability and accuracy of NGS results.

Implementation Method 1

a double-stranded nucleic acid molecule comprising a sense strand containing a type IIs restriction enzyme recognition site and a random nucleotide sequence of a length of 3 to 30 nucleotides (nt); and an anti-sense strand comprising a complementary sequence to the sense strand and a 3′-end A tail

Methodology Applied

Scientific EffectBase pairing:

Implementation Method 2

treating the resulting reaction products with a type IIs restriction enzyme

Methodology Applied

Scientific EffectRestriction enzyme cutting: Enzyme

Implementation Method 3

treating and reacting a double-stranded nucleic acid molecule according to the present invention and DNA ligase in a DNA library

Methodology Applied

Scientific EffectDNA ligation:

Data Source

PatentUS20230059086A1Double-stranded nucleic acid molecules and method for removing glass adaptor in DNA library by means of same

Publication Date: 2023.02.23 EONEDIAGNOMICS CO LTD

AI summary

The present invention relates to a method for removing free adapters in a DNA library using a double-stranded nucleic acid molecule and a restriction enzyme, and more specifically, to a method for removing free adapters in a DNA library for next generation sequencing (NGS) using a double-stranded nucleic acid molecule including a type IIs restriction enzyme recognition site and a complementary sequence thereof, and a type IIs restriction enzyme.