Complete sequence verification of plasmid DNA using the Oxford Nanopore Technologies’ MinION device

Abstract

Background Sequence verification is essential for plasmids used as critical reagents or therapeutic products. Typically, high-quality plasmid sequence is achieved through capillary-based Sanger sequencing, requiring customized sets of primers for each plasmid. This process can become expensive, particularly for applications where the validated sequence needs to be produced within a regulated and quality-controlled environment for downstream clinical research applications. Results Here, we describe a cost-effective and accurate plasmid sequencing and consensus generation procedure using the Oxford Nanopore Technologies’ MinION device as an alternative to capillary-based plasmid sequencing options. This procedure can verify the identity of a pure population of plasmid, either confirming it matches the known and expected sequence, or identifying mutations present in the plasmid if any exist. We use a full MinION flow cell per plasmid, maximizing available data and allowing for stringent quality filters. Pseudopairing reads for consensus base calling reduces read error rates from 5.3 to 0.53%, and our pileup consensus approach provides per-base counts and confidence scores, allowing for interpretation of the certainty of the resulting consensus sequences. For pure plasmid samples, we demonstrate 100% accuracy in the resulting consensus sequence, and the sensitivity to detect small mutations such as insertions, deletions, and single nucleotide variants. In test cases where the sequenced pool of plasmids contains subclonal templates, detection sensitivity is similar to that of traditional capillary sequencing. Conclusions Our pipeline can provide significant cost savings compared to outsourcing clinical-grade sequencing of plasmids, making generation of high-quality plasmid sequence for clinical sequence verification more accessible. While other long-read-based methods offer higher-throughput and less cost, our pipeline produces complete and accurate sequence verification for cases where absolute sequence accuracy is required.

Biofoundry-Scale DNA Assembly Validation Using Cost-Effective High-Throughput Long-Read Sequencing

“…The SequenceGenie workflow analyzes data from a multiplexed sequencing approach using a novel sample barcoding system . The MinION Plasmid Sequence Verification Pipeline provides a cost-effective way of sequencing plasmids for clinical research applications . Circuit-seq creates de novo assemblies from multiplexed samples, while OnRamp is reference-based .…”

“… 12 The MinION Plasmid Sequence Verification Pipeline provides a cost-effective way of sequencing plasmids for clinical research applications. 13 Circuit-seq creates de novo assemblies from multiplexed samples, 14 while OnRamp is reference-based. 15 In comparison to these, Sequeduct performs an evaluation against an expected sequence and focuses on the produced report and downstream interpretation of results, which are more suitable for engineering biology and quality control purposes.…”

Biofoundry-scale DNA assembly validation using cost-effective high-throughput long read sequencing

“…The SequenceGenie workflow analyses data from a multiplexed sequencing approach using a novel sample barcoding system 11 . The MinION Plasmid Sequence Verification Pipeline provides a cost-effective way of sequencing plasmids for clinical research applications 12 . Circuit-seq creates de novo assemblies from multiplexed samples 13 while OnRamp is reference-based 14 .…”

Investigation of multidrug-resistant plasmids from carbapenemase-producing Klebsiella pneumoniae clinical isolates from Pakistan

“…; abundance of repetitive elements), and offers a potential solution in real-time and at a much lower cost (e.g., ~$2 K for a start-up kit) for reconstructing MGEs (e.g. ; plasmids) and facilitating the identification of ARGs while also helping to track plasmid dissemination and the spread of AMR ( Brown et al, 2023 ).…”