RT Journal Article SR Electronic T1 Circuit-seq: Circular reconstruction of cut in vitro transposed plasmids using Nanopore sequencing JF bioRxiv FD Cold Spring Harbor Laboratory SP 2022.01.25.477550 DO 10.1101/2022.01.25.477550 A1 Francesco E. Emiliani A1 Ian Hsu A1 Aaron McKenna YR 2022 UL http://biorxiv.org/content/early/2022/01/26/2022.01.25.477550.abstract AB Recombinant DNA is a fundamental tool in biotechnology and medicine. Validation of the resulting plasmid sequence 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 for approximately $1.50 per plasmid. We demonstrate the power of Circuit-seq across a wide range of plasmid sizes and complexities, generating accurate and 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 cost than commercial Sanger sequencing, accelerating a key step in synthetic biology, with low startup costs make it practical for individual laboratories.Competing Interest StatementThe authors have declared no competing interest.