DuBA.flow─A low-cost, long-read amplicon sequencing workflow for the validation 
of synthetic DNA constructs

Ramírez Rojas, Adán Andrés; Brinkmann, Cedric K.; Köbel, Tania S.; Schindler, Daniel

doi:10.1021/acssynbio.3c00522

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DuBA.flow─A low-cost, long-read amplicon sequencing workflow for the validation of synthetic DNA constructs

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Ramírez Rojas, Adán Andrés
Core Facility MPG MAXGenesys DNAfoundry, Max Planck Institute for Terrestrial Microbiology, Max Planck Society;

Brinkmann, Cedric K.
Core Facility MPG MAXGenesys DNAfoundry, Max Planck Institute for Terrestrial Microbiology, Max Planck Society;

Köbel, Tania S.
Core Facility MPG MAXGenesys DNAfoundry, Max Planck Institute for Terrestrial Microbiology, Max Planck Society;

/persons/resource/persons261244

Schindler, Daniel
Core Facility MPG MAXGenesys DNAfoundry, Max Planck Institute for Terrestrial Microbiology, Max Planck Society;

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https://doi.org/10.1021/acssynbio.3c00522
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Citation

Ramírez Rojas, A. A., Brinkmann, C. K., Köbel, T. S., & Schindler, D. (2024). DuBA.flow─A low-cost, long-read amplicon sequencing workflow for the validation of synthetic DNA constructs. ACS Synthetic Biology. doi:10.1021/acssynbio.3c00522.

Cite as: https://hdl.handle.net/21.11116/0000-000E-58E9-9

Abstract

Modern biological science, especially synthetic biology, relies heavily on the construction of DNA elements, often in the form of plasmids. Plasmids are used for a variety of applications, including the expression of proteins for subsequent purification, the expression of heterologous pathways for the production of valuable compounds, and the study of biological functions and mechanisms. For all applications, a critical step after the construction of a plasmid is its sequence validation. The traditional method for sequence determination is Sanger sequencing, which is limited to approximately 1000 bp per reaction. Here, we present a highly scalable in-house method for rapid validation of amplified DNA sequences using long-read Nanopore sequencing. We developed two-step amplicon and transposase strategies to provide maximum flexibility for dual barcode sequencing. We also provide an automated analysis pipeline to quickly and reliably analyze sequencing results and provide easy-to-interpret results for each sample. The user-friendly DuBA.flow start-to-finish pipeline is widely applicable. Furthermore, we show that construct validation using DuBA.flow can be performed by barcoded colony PCR amplicon sequencing, thus accelerating research.