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Sequencing Strategy to Ensure Accurate Plasmid Assembly
Despite the wide use of plasmids in research and clinical production, the need to verify plasmid sequences is a bottleneck that is too often underestimated in the manufacturing process. Although sequencing platforms continue to improve, the method and assembly pipeline chosen still influence the fin...
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Published in: | ACS synthetic biology 2024-12, Vol.13 (12), p.4099 |
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creator | Hernandez, Sarah I Berezin, Casey-Tyler Miller, Katie M Peccoud, Samuel J Peccoud, Jean |
description | Despite the wide use of plasmids in research and clinical production, the need to verify plasmid sequences is a bottleneck that is too often underestimated in the manufacturing process. Although sequencing platforms continue to improve, the method and assembly pipeline chosen still influence the final plasmid assembly sequence. Furthermore, few dedicated tools exist for plasmid assembly, especially for
assembly. Here, we evaluated short-read, long-read, and hybrid (both short and long reads)
assembly pipelines across three replicates of a 24-plasmid library. Consistent with previous characterizations of each sequencing technology, short-read assemblies had issues resolving GC-rich regions, and long-read assemblies commonly had small insertions and deletions, especially in repetitive regions. The hybrid approach facilitated the most accurate, consistent assembly generation and identified mutations relative to the reference sequence. Although Sanger sequencing can be used to verify specific regions, some GC-rich and repetitive regions were difficult to resolve using any method, suggesting that easily sequenced genetic parts should be prioritized in the design of new genetic constructs. |
doi_str_mv | 10.1021/acssynbio.4c00539 |
format | article |
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assembly. Here, we evaluated short-read, long-read, and hybrid (both short and long reads)
assembly pipelines across three replicates of a 24-plasmid library. Consistent with previous characterizations of each sequencing technology, short-read assemblies had issues resolving GC-rich regions, and long-read assemblies commonly had small insertions and deletions, especially in repetitive regions. The hybrid approach facilitated the most accurate, consistent assembly generation and identified mutations relative to the reference sequence. Although Sanger sequencing can be used to verify specific regions, some GC-rich and repetitive regions were difficult to resolve using any method, suggesting that easily sequenced genetic parts should be prioritized in the design of new genetic constructs.</description><identifier>ISSN: 2161-5063</identifier><identifier>EISSN: 2161-5063</identifier><identifier>DOI: 10.1021/acssynbio.4c00539</identifier><identifier>PMID: 39508818</identifier><language>eng</language><publisher>United States</publisher><subject>Escherichia coli - genetics ; Gene Library ; High-Throughput Nucleotide Sequencing - methods ; Plasmids - genetics ; Sequence Analysis, DNA - methods</subject><ispartof>ACS synthetic biology, 2024-12, Vol.13 (12), p.4099</ispartof><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><cites>FETCH-LOGICAL-c183t-6fd87c60eeaa90e6e15c82477ef4888b02d7923934c7ccfc47e26c3d665699ab3</cites><orcidid>0000-0001-7649-6127</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/39508818$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Hernandez, Sarah I</creatorcontrib><creatorcontrib>Berezin, Casey-Tyler</creatorcontrib><creatorcontrib>Miller, Katie M</creatorcontrib><creatorcontrib>Peccoud, Samuel J</creatorcontrib><creatorcontrib>Peccoud, Jean</creatorcontrib><title>Sequencing Strategy to Ensure Accurate Plasmid Assembly</title><title>ACS synthetic biology</title><addtitle>ACS Synth Biol</addtitle><description>Despite the wide use of plasmids in research and clinical production, the need to verify plasmid sequences is a bottleneck that is too often underestimated in the manufacturing process. Although sequencing platforms continue to improve, the method and assembly pipeline chosen still influence the final plasmid assembly sequence. Furthermore, few dedicated tools exist for plasmid assembly, especially for
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assembly. Here, we evaluated short-read, long-read, and hybrid (both short and long reads)
assembly pipelines across three replicates of a 24-plasmid library. Consistent with previous characterizations of each sequencing technology, short-read assemblies had issues resolving GC-rich regions, and long-read assemblies commonly had small insertions and deletions, especially in repetitive regions. The hybrid approach facilitated the most accurate, consistent assembly generation and identified mutations relative to the reference sequence. Although Sanger sequencing can be used to verify specific regions, some GC-rich and repetitive regions were difficult to resolve using any method, suggesting that easily sequenced genetic parts should be prioritized in the design of new genetic constructs.</abstract><cop>United States</cop><pmid>39508818</pmid><doi>10.1021/acssynbio.4c00539</doi><orcidid>https://orcid.org/0000-0001-7649-6127</orcidid></addata></record> |
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source | American Chemical Society:Jisc Collections:American Chemical Society Read & Publish Agreement 2022-2024 (Reading list) |
subjects | Escherichia coli - genetics Gene Library High-Throughput Nucleotide Sequencing - methods Plasmids - genetics Sequence Analysis, DNA - methods |
title | Sequencing Strategy to Ensure Accurate Plasmid Assembly |
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