Barcode-free multiplex plasmid sequencing using Bayesian analysis and nanopore sequencing
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 o...
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eLife Sciences Publications Ltd
2025-04-01
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| Online Access: | https://elifesciences.org/articles/88794 |
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| author | Masaaki Uematsu Jeremy M Baskin |
| author_facet | Masaaki Uematsu Jeremy M Baskin |
| author_sort | Masaaki Uematsu |
| collection | DOAJ |
| description | 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 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. |
| format | Article |
| id | doaj-art-4d1c3ffc43544e5ebbe2a8dd49eee777 |
| institution | DOAJ |
| issn | 2050-084X |
| language | English |
| publishDate | 2025-04-01 |
| publisher | eLife Sciences Publications Ltd |
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| series | eLife |
| spelling | doaj-art-4d1c3ffc43544e5ebbe2a8dd49eee7772025-08-20T03:14:19ZengeLife Sciences Publications LtdeLife2050-084X2025-04-011210.7554/eLife.88794Barcode-free multiplex plasmid sequencing using Bayesian analysis and nanopore sequencingMasaaki Uematsu0https://orcid.org/0000-0002-0197-8401Jeremy M Baskin1https://orcid.org/0000-0003-2939-3138Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, United StatesWeill Institute for Cell and Molecular Biology, Cornell University, Ithaca, United States; Department of Chemistry and Chemical Biology, Cornell University, Ithaca, United StatesPlasmid 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 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.https://elifesciences.org/articles/88794nanopore sequencingBayesian analysiswhole-plasmid sequencingplasmid sequencing |
| spellingShingle | Masaaki Uematsu Jeremy M Baskin Barcode-free multiplex plasmid sequencing using Bayesian analysis and nanopore sequencing eLife nanopore sequencing Bayesian analysis whole-plasmid sequencing plasmid sequencing |
| title | Barcode-free multiplex plasmid sequencing using Bayesian analysis and nanopore sequencing |
| title_full | Barcode-free multiplex plasmid sequencing using Bayesian analysis and nanopore sequencing |
| title_fullStr | Barcode-free multiplex plasmid sequencing using Bayesian analysis and nanopore sequencing |
| title_full_unstemmed | Barcode-free multiplex plasmid sequencing using Bayesian analysis and nanopore sequencing |
| title_short | Barcode-free multiplex plasmid sequencing using Bayesian analysis and nanopore sequencing |
| title_sort | barcode free multiplex plasmid sequencing using bayesian analysis and nanopore sequencing |
| topic | nanopore sequencing Bayesian analysis whole-plasmid sequencing plasmid sequencing |
| url | https://elifesciences.org/articles/88794 |
| work_keys_str_mv | AT masaakiuematsu barcodefreemultiplexplasmidsequencingusingbayesiananalysisandnanoporesequencing AT jeremymbaskin barcodefreemultiplexplasmidsequencingusingbayesiananalysisandnanoporesequencing |