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Validating Whole Genome Nanopore Sequencing, using Usutu Virus as an Example

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Published on: March 11, 2020

Validation of Golden Gate Assemblies Using Highly Multiplexed Nanopore Amplicon Sequencing.

Adán A Ramírez Rojas¹, Cedric K Brinkmann¹, Daniel Schindler^2,3

¹Max Planck Institute for Terrestrial Microbiology, Marburg, Germany.

Methods in Molecular Biology (Clifton, N.J.)

|October 3, 2024

Summary

Golden Gate cloning enables complex biological engineering. A new dual barcode amplicon sequencing workflow (DuBA.flow) on Nanopore platforms streamlines in-house validation for complex assemblies, simplifying synthetic biology research.

Keywords:

Colony PCR DNA assembly validation Dual-barcode amplicon sequencing Enzymatic fragmentation Golden Gate assembly validation Laboratory automation Nanopore sequencing amplicon sequencing

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Last Updated: Jun 11, 2025

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Area of Science:

Synthetic Biology
Molecular Biology
Genomics

Background:

Golden Gate cloning facilitates modular assembly of DNA parts for biological engineering.
While defined parts allow simple validation, complex assemblies from part libraries necessitate robust sequencing.
Current validation methods can be insufficient for high-complexity constructs.

Purpose of the Study:

To present a detailed protocol for in-house sequence validation of complex DNA assemblies.
To introduce a highly multiplexed dual barcode amplicon sequencing workflow (DuBA.flow) for Nanopore sequencing.
To provide a start-to-finish solution for validating Golden Gate assemblies.

Main Methods:

Development of a multiplexed dual barcode amplicon sequencing strategy.
Adaptation of the workflow for the Nanopore sequencing platform.
Creation of a comprehensive protocol from colony to sequencing report.

Main Results:

DuBA.flow enables efficient, in-house sequence validation of complex Golden Gate assemblies.
The workflow integrates seamlessly with Nanopore sequencing technology.
A user-friendly, easy-to-interpret sequencing report is generated.

Conclusions:

DuBA.flow offers a powerful solution for validating complex synthetic biology constructs.
This method enhances the reliability and efficiency of engineering biological systems.
In-house Nanopore sequencing validation is feasible and beneficial for complex cloning projects.