Bubble-Shaped Adaptor Element for Sequencing Library Construction

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Solution Overview

Problem

The conventional process for constructing a whole genome library using the Complete Genomics sequencing platform is time-consuming and costly due to the cumbersome steps involved in ligating adaptors.

Innovation Solution

A method is introduced that uses bubble-shaped adaptors to reduce the number of ligating steps and PCR amplification cycles, optimizing library construction by ligating two bubble-shaped adaptors with the same sequences in one step, allowing for direct PCR amplification without intermediate processes.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If conventional two-set adaptors are used for library construction, then complete sequencing coverage is achieved, but the process becomes time-consuming and complex

Engineering Contradiction:

Improvesequencing coverageVSAvoidlibrary construction time

Core Design Contradiction:

ReliabilityVSLoss of time

Solution Approach 1:

The patent merges the functions of two separate adaptors into a single adaptor structure containing both forward and reverse sequencing regions. This single adaptor performs multiple functions that previously required two separate adaptor ligation steps, thereby reducing construction time while maintaining complete sequencing coverage.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The designed adaptor serves multiple purposes: it provides both forward and reverse sequencing priming sites, enables single-step ligation, and maintains compatibility with the sequencing platform. This multi-functional adaptor eliminates the need for separate adaptors for different sequencing directions.

Inventive Principle:

Principle #6Universality (Multi-functionality)

2Reliability

If multiple ligating steps are performed, then adaptors are properly attached to DNA fragments, but the overall process becomes cumbersome and costly

Engineering Contradiction:

Improveadaptor ligation efficiencyVSAvoidlibrary construction steps

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The patent combines multiple adaptor ligation operations into a single ligation step by using a multifunctional adaptor design. This reduces the number of procedural steps from multiple ligation reactions to one, simplifying the workflow while ensuring proper adaptor attachment to DNA fragments.

Inventive Principle:

Principle #5Merging (Combining)

3Reliability

If conventional adaptor ligation process is used, then sequencing libraries are constructed, but material costs and time consumption increase

Engineering Contradiction:

Improvelibrary construction qualityVSAvoidlibrary construction efficiency

Core Design Contradiction:

ReliabilityVSProductivity

Solution Approach 1:

The patent merges multiple construction steps into a streamlined single-step ligation process using the designed adaptor, thereby improving productivity and reducing material consumption without compromising the quality of the constructed sequencing libraries.

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

This approach significantly reduces the time and material costs associated with library construction, improving efficiency by simplifying the process and enabling direct PCR amplification of the adaptor-ligated product.

Implementation Method 1

the adaptor element 1 includes a long-chain nucleic acid and a short-chain nucleic acid, which are capable of being annealed to form a first hybrid in a bubble shape

Methodology Applied

Scientific EffectAnnealing: Annealing

Implementation Method 2

the first pair of primers each contains a site in the middle thereof, preferably Uracil (U) or deoxyuridine (dU), recognizable and digestible by an enzyme, preferably a USER enzyme

Methodology Applied

Scientific EffectEnzyme digestion: Enzyme

Implementation Method 3

the adaptor element 1 contains a recognition site of class III restriction endonuclease

Methodology Applied

Scientific EffectRestriction enzyme digestion: Enzyme

Implementation Method 4

ligating an adaptor element 1 to both ends of the DNA fragment obtained in step 1)

Methodology Applied

Scientific EffectLigation:

Implementation Method 5

phosphorylation at the 5′-terminal

Methodology Applied

Scientific EffectPhosphorylation:

Implementation Method 6

adenine (A) addition to the 3′-terminal

Methodology Applied

Scientific EffectAdenine addition:

Implementation Method 7

subjecting a double stranded DNA fragment obtained to end-repairing

Methodology Applied

Scientific EffectEnd-repairing:

Implementation Method 8

capturing the PCR product obtained in step 8) with avidin-coated magnetic beads

Methodology Applied

Scientific EffectBiotin-avidin binding: Adsorption

Data Source

PatentUS10316356B1Method of constructing sequencing library with bubble-shaped adaptor element

Publication Date: 2019.06.11 MGI TECH CO LTD

AI summary

Provided is a method of constructing a sequencing library using an adaptor element in a bubble shape. The bubble-shaped adaptor is ligated to a DNA fragment respectively at the 3′-terminal and the 5′-terminal i.e., two bubble-shaped adaptors with same sequences are ligated in one step. The bubble-shaped adaptor-ligated product is then amplified with a primer complementary to the 3′-terminal of the long-chain nucleic acid of the bubble-shaped adaptor, so as to replace the non-paired sequence in the short-chain nucleic acid.