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bioRxiv · 10.1101/2023.04.12.536413

Barcode-free multiplex plasmid sequencing using Bayesian analysis and nanopore sequencing

Uematsu, M.·Baskin, J. M.
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Abstract

Plasmid construction is central to life science research, and sequence verification is arguably its costliest step. Long-read sequencing has emerged as a competitor to Sanger sequencing, with the principal benefit that whole plasmids can be sequenced in a single run. Nevertheless, the current cost of nanopore sequencing is still prohibitive for routine sequencing during plasmid construction. 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 mixtures in a pre-survey step, and following sequencing, executes a post-analysis workflow involving sequence classification, alignment, and consensus determination. By using Bayesian analysis with prior probability of expected plasmid construction error rate, high-confidence sequences can be obtained for each plasmid in the mixture. Plasmids differing by as little as two bases can be mixed for submission as a single sample for nanopore sequencing, and routine multiplexing of even six plasmids per 180 reads can still maintain high accuracy of consensus sequencing. 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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Uematsu, M., Baskin, J. M.. 2023-04-13. Barcode-free multiplex plasmid sequencing using Bayesian analysis and nanopore sequencing. https://doi.org/10.1101/2023.04.12.536413

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