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

Abstract

Sequence verification is essential for plasmids used as critical reagents or therapeutic products. 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 commercial 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 basecalling reduces error rates to 0.53 %, and our pileup consensus approach provides per-base counts and confidence scores, allowing for interpretation 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 cases where the sequenced pool of plasmids contains subclonal templates, detection sensitivity is similar to that of traditional capillary sequencing. 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.

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 .…”

OnRamp: rapid nanopore plasmid validation

“…OnRamp uses a reference-based system for analysis, as it is a tool designed specifically for routine plasmid validation. While this precludes use of OnRamp for de novo assembly of unknown plasmid sequences (an uncommon case in routine screening), there already exist well-designed tools for de novo assembly of nanopore data (Emiliani et al 2022; Currin et al 2019; Brown et al 2022). For instance, while we were able to detect most variation in our constructs, consensus sequences for plasmids with very large indels (>1000bp) or where large portions of the plasmid have inserted backwards relative to the reference could not be generated.…”

“…DNA sequencing using nanopore requires ligation of DNA ends with specialized adapters used to facilitate sequencing. While some have previously utilized ONT for plasmid validation, our method is unique in that it leverages full-length plasmid reads for assembly and does not require barcodes which allows for a rapid and simple sample preparation (Emiliani et al 2022;Currin et al 2019;Brown et al 2022). Here we provide two methods for plasmid pool preparation for OnRamp runs based on the adapter ligation method: transposase-based or restriction digest-based (Fig.…”

Sequencing Strategy to Ensure Accurate Plasmid Assembly

“…However, many of these methods can struggle to accurately reconstruct small (<25 kbp) plasmids or miss them altogether [26, 27]. To accelerate plasmid verification in high- throughput settings, assembly pipelines should aim to avoid the need for a reference sequence or manual intervention, as required by some methods [3, 28]. Here we used the two major end-to-end de novo assembly pipelines designed for plasmids: Epi2ME, a tool from Oxford Nanopore Technologies (ONT) for long-read assembly, and the open-source plasmid tool, PlasCAT, which can perform short-read, long-read, and hybrid assembly [2, 29–31].…”