Scalable Combinatorial Assembly of Synthetic DNA for Tracking Applications

• Published in: International journal of molecular sciences Vol. 24; no. 3; p. 2549
• Main Authors: Stuart, Julius D, Wickenkamp, Natalie R, Davis, Kaleb A, Meyer, Camden, Kading, Rebekah C, Snow, Christopher D
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 29.01.2023; MDPI
• Subjects: Combinatorial analysis; combinatorial library; computational design; Contamination; Datasets; Deoxyribonucleic acid; Design; DNA; DNA - analysis; DNA - genetics; DNA assembly; DNA barcoding; DNA Barcoding, Taxonomic - methods; Gene Library; High-Throughput Nucleotide Sequencing - methods; Information storage; Modular construction; Modular design; Multiplexing; Next-generation sequencing; Nucleotide sequence; Oligonucleotides - genetics; Quantum dots; Sequence Analysis, DNA - methods; surveillance; synthetic DNA barcoding; synthetic DNA tags; Universal Product Code; next-generation sequencing; computational design; surveillance; synthetic DNA barcoding; DNA assembly; combinatorial library; synthetic DNA tags
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms24032549

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.