[Paper Review] Validation of Golden Gate assemblies using highly multiplexed Nanopore amplicon sequencing

Adán Andrés Ramírez Rojas, Cedric K. Brinkmann+1 more|arXiv (Cornell University)|Jan 25, 2024

Genomics and Phylogenetic StudiesBiochemistry, Genetics and Molecular Biology3 citations

TL;DR

This paper presents a highly multiplexed, dual-barcode amplicon sequencing workflow using Oxford Nanopore sequencing to validate complex Golden Gate assemblies, particularly when using part libraries rather than defined, validated parts. The method enables comprehensive, cost-effective, and high-throughput validation of combinatorial Golden Gate constructs directly from single colonies, delivering accurate, interpretable sequencing reports with minimal hands-on time and high sensitivity to sequence errors or chimeras.

ABSTRACT

Golden Gate cloning has revolutionized synthetic biology. Its concept of modular, highly characterized libraries of parts that can be combined into higher order assemblies allows engineering principles to be applied to biological systems. The basic parts, typically stored in level 0 plasmids, are sequence validated by the method of choice and can be combined into higher order assemblies on demand. Higher order assemblies are typically transcriptional units, and multiple transcriptional units can be assembled into multi-gene constructs. Higher order Golden Gate assembly based on defined and validated parts usually does not introduce sequence changes. Therefore, simple validation of the assemblies, e.g. by colony PCR or restriction digest pattern analysis, is sufficient. However, in many experimental setups, researchers do not use defined parts, but rather part libraries, resulting in assemblies of high combinatorial complexity where sequencing again becomes mandatory. Here we present a detailed protocol for the use of a highly multiplexed dual barcode amplicon sequencing using the Nanopore sequencing platform for in-house sequence validation. The workflow, called DuBA.flow, is a start-to-finish procedure that provides all necessary steps from a single colony to the final easy-to-interpret sequencing report.

Motivation & Objective

• To address the challenge of validating high-complexity Golden Gate assemblies generated from part libraries rather than defined, sequence-verified parts.
• To develop a streamlined, end-to-end workflow for in-house validation of Golden Gate assemblies using Nanopore sequencing.
• To enable high-throughput, cost-effective, and accurate detection of sequence errors, chimeras, or unintended rearrangements in combinatorial assemblies.
• To provide a user-friendly, easy-to-interpret sequencing report directly from single colonies without reliance on Sanger sequencing or low-complexity methods.

Proposed method

• Utilizes highly multiplexed amplicon sequencing with dual barcodes to uniquely tag individual colonies and their PCR amplicons.
• Employs Oxford Nanopore sequencing technology to generate long-read, high-accuracy sequencing data suitable for detecting complex assembly errors.
• Applies a custom bioinformatics pipeline to demultiplex samples, map reads to expected constructs, and identify sequence variants, chimeras, or mutations.
• Integrates a single-colony-to-report workflow that minimizes manual steps and enables rapid validation of combinatorial Golden Gate assemblies.
• Uses dual barcoding to prevent sample cross-talk and improve multiplexing capacity and data reliability.
• Produces a final report that clearly indicates the identity and fidelity of each assembled construct, including error detection and variant calling.

Experimental results

Research questions

• RQ1 Can a highly multiplexed, dual-barcode amplicon sequencing approach using Nanopore sequencing reliably validate complex Golden Gate assemblies from part libraries?
• RQ2 How does this method compare to traditional validation techniques like Sanger sequencing or restriction digest in terms of throughput, accuracy, and error detection?
• RQ3 To what extent can this workflow detect chimeric constructs, point mutations, or incorrect part fusions in combinatorial assemblies?
• RQ4 Can the method be implemented in a standard molecular biology lab with minimal bioinformatics expertise and without external sequencing services?

Key findings

• The method enables the simultaneous validation of hundreds of Golden Gate assemblies in a single sequencing run, significantly increasing throughput compared to Sanger sequencing.
• The dual-barcode strategy effectively prevents sample cross-talk and enables accurate demultiplexing even at high multiplexing depths.
• The workflow successfully detected sequence errors, chimeric fusions, and incorrect part combinations in complex assemblies that would have been missed by colony PCR or restriction digest alone.
• The final sequencing report is highly interpretable, providing clear, actionable data on construct identity and fidelity directly from single colonies.
• The entire workflow—from colony picking to final report—can be completed in a single lab with minimal hands-on time and standard equipment.
• The method demonstrated high sensitivity to low-abundance errors and chimeras, making it suitable for high-fidelity synthetic biology applications.