Multiplexed Assembly and Annotation of Synthetic Biology Constructs Using Long-Read Nanopore Sequencing

作者：Francesco E. Emiliani, Ian Hsu, Aaron McKenna · 发表于：ACS Synthetic Biology · 年份：2022 · DOI：10.1021/acssynbio.2c00126 · 被引用次数：29 · 研究领域：Genomics and Phylogenetic Studies、Advanced biosensing and bioanalysis techniques、Single-cell and spatial transcriptomics

Recombinant DNA is a fundamental tool in biotechnology and medicine. These DNA sequences are often built, replicated, and delivered in the form of plasmids. Validation of these plasmid sequences is a critical and time-consuming step, which has been dominated for the last 35 years by Sanger sequencing. As plasmid sequences grow more complex with new DNA synthesis and cloning techniques, we need new approaches that address the corresponding validation challenges at scale. Here we prototype a high-throughput plasmid sequencing approach using DNA transposition and Oxford Nanopore sequencing. Our method, Circuit-seq, creates robust, full-length, and accurate plasmid assemblies without prior knowledge of the underlying sequence. We demonstrate the power of Circuit-seq across a wide range of plasmid sizes and complexities, generating full-length, contiguous plasmid maps. We then leverage our long-read data to characterize epigenetic marks and estimate plasmid contamination levels. Circuit-seq scales to large numbers of samples at a lower per-sample cost than commercial Sanger sequencing, accelerating a key step in synthetic biology, while low equipment costs make it practical for individual laboratories.