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Scalable Combinatorial Assembly of Synthetic DNA for Tracking Applications

Synthetic DNA barcodes are double-stranded DNA molecules designed to carry recoverable information, information that can be used to represent and track objects and organisms. DNA barcodes offer robust, sensitive detection using standard amplification and sequencing techniques. While numerous researc...

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Autores principales: Stuart, Julius D., Wickenkamp, Natalie R., Davis, Kaleb A., Meyer, Camden, Kading, Rebekah C., Snow, Christopher D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9917336/
https://www.ncbi.nlm.nih.gov/pubmed/36768872
http://dx.doi.org/10.3390/ijms24032549
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author Stuart, Julius D.
Wickenkamp, Natalie R.
Davis, Kaleb A.
Meyer, Camden
Kading, Rebekah C.
Snow, Christopher D.
author_facet Stuart, Julius D.
Wickenkamp, Natalie R.
Davis, Kaleb A.
Meyer, Camden
Kading, Rebekah C.
Snow, Christopher D.
author_sort Stuart, Julius D.
collection PubMed
description Synthetic DNA barcodes are double-stranded DNA molecules designed to carry recoverable information, information that can be used to represent and track objects and organisms. DNA barcodes offer robust, sensitive detection using standard amplification and sequencing techniques. While numerous research groups have promoted DNA as an information storage medium, less attention has been devoted to the design of economical, scalable DNA barcode libraries. Here, we present an alternative modular approach to sequence design. Barcode sequences were constructed from smaller, interchangeable blocks, allowing for the combinatorial assembly of numerous distinct tags. We demonstrated the design and construction of first-generation (N = 256) and second-generation (N = 512) modular barcode libraries, from fewer than 50 total single-stranded oligonucleotides for each library. To avoid contamination during experimental validation, a liquid-handling robot was employed for oligonucleotide mixing. Generating barcode sequences in-house reduces dependency upon external entities for unique tag generation, increasing flexibility in barcode generation and deployment. Next generation sequencing (NGS) detection of 256 different samples in parallel highlights the multiplexing afforded by the modular barcode design coupled with high-throughput sequencing. Deletion variant analysis of the first-generation library informed sequence design for enhancing barcode assembly specificity in the second-generation library.
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spelling pubmed-99173362023-02-11 Scalable Combinatorial Assembly of Synthetic DNA for Tracking Applications Stuart, Julius D. Wickenkamp, Natalie R. Davis, Kaleb A. Meyer, Camden Kading, Rebekah C. Snow, Christopher D. Int J Mol Sci Article Synthetic DNA barcodes are double-stranded DNA molecules designed to carry recoverable information, information that can be used to represent and track objects and organisms. DNA barcodes offer robust, sensitive detection using standard amplification and sequencing techniques. While numerous research groups have promoted DNA as an information storage medium, less attention has been devoted to the design of economical, scalable DNA barcode libraries. Here, we present an alternative modular approach to sequence design. Barcode sequences were constructed from smaller, interchangeable blocks, allowing for the combinatorial assembly of numerous distinct tags. We demonstrated the design and construction of first-generation (N = 256) and second-generation (N = 512) modular barcode libraries, from fewer than 50 total single-stranded oligonucleotides for each library. To avoid contamination during experimental validation, a liquid-handling robot was employed for oligonucleotide mixing. Generating barcode sequences in-house reduces dependency upon external entities for unique tag generation, increasing flexibility in barcode generation and deployment. Next generation sequencing (NGS) detection of 256 different samples in parallel highlights the multiplexing afforded by the modular barcode design coupled with high-throughput sequencing. Deletion variant analysis of the first-generation library informed sequence design for enhancing barcode assembly specificity in the second-generation library. MDPI 2023-01-29 /pmc/articles/PMC9917336/ /pubmed/36768872 http://dx.doi.org/10.3390/ijms24032549 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Stuart, Julius D.
Wickenkamp, Natalie R.
Davis, Kaleb A.
Meyer, Camden
Kading, Rebekah C.
Snow, Christopher D.
Scalable Combinatorial Assembly of Synthetic DNA for Tracking Applications
title Scalable Combinatorial Assembly of Synthetic DNA for Tracking Applications
title_full Scalable Combinatorial Assembly of Synthetic DNA for Tracking Applications
title_fullStr Scalable Combinatorial Assembly of Synthetic DNA for Tracking Applications
title_full_unstemmed Scalable Combinatorial Assembly of Synthetic DNA for Tracking Applications
title_short Scalable Combinatorial Assembly of Synthetic DNA for Tracking Applications
title_sort scalable combinatorial assembly of synthetic dna for tracking applications
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9917336/
https://www.ncbi.nlm.nih.gov/pubmed/36768872
http://dx.doi.org/10.3390/ijms24032549
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