Barcode-free multiplex plasmid sequencing using Bayesian analysis and nanopore sequencing
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.12.536413v1?rss=1

Authors: Uematsu, M., Baskin, J. M.

Abstract: Plasmid construction is central to molecular life science research, and sequence verification is arguably the costliest step in the process. Long-read sequencing has recently emerged as competitor to Sanger sequencing, with the principal benefit that whole plasmids can be sequenced in a single run. Though nanopore and related long-read technologies feature lower base-calling accuracies, high-quality sequencing can be achieved by obtaining a consensus from multiple reads. Nevertheless, the current cost of nanopore sequencing is still prohibitive for routine sequencing during plasmid construction. Here, we develop a computational approach termed Simple Algorithm for Very Efficient Multiplexing of Oxford Nanopore Experiments for You (SAVEMONEY) that guides researchers to mix multiple plasmids and subsequently computationally de-mixes the resultant sequences. SAVEMONEY defines optimal plasmid mixtures in a pre-survey step, and following sequencing, executes a three-part post-analysis workflow involving sequence classification, alignment, and consensus determination steps. By using Bayesian analysis with prior probability of expected plasmid construction error rate in the consensus determination step, high-confidence sequences can be obtained for each plasmid in the mixture. Critically, we demonstrate that plasmids differing by as little as two bases can be mixed for submission as a single sample for nanopore sequencing, and routine multiplexing of six plasmids in a single sample can still yield highly accurate sequencing information. SAVEMONEY should further democratize whole-plasmid sequencing by nanopore and related technologies, driving down the effective cost of whole-plasmid sequencing to lower than that of a single Sanger sequencing run.

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