Hybrid Sequencing Facilitates Robust De Novo Plasmid Assembly.
Hybrid Sequencing Facilitates Robust De Novo Plasmid Assembly.
复制标题
混合测序有利于稳健的从头组装质粒。
DOI：
10.1101/2024.03.25.586694
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发表时间：
2024
期刊：
影响因子：
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通讯作者：
Peccoud,Jean
中科院分区：
文献类型：
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作者：
Hernandez,SarahI;Berezin,Casey-Tyler;Miller,KatieM;Peccoud,SamuelJ;Peccoud,Jean
尽管质粒在研究和临床生产中广泛使用，但质粒序列的验证是制造过程中经常被忽视的瓶颈。虽然测序平台不断改进，但选择的方法和组装管道仍然影响最终的质粒组装序列。此外，很少有专门用于质粒组装的工具，特别是用于新组装的工具。在这里，我们评估了短读、长读和混合（包括短读和长读）在24个质粒库的三个复制中的新组装管道。与之前每种测序技术的特征一致，短读序列在解析富含gc的区域时经常出现问题，而长读序列通常有小的插入和缺失，特别是在重复区域。混合方法促进了最一致的装配生成。尽管Sanger测序可用于验证特定区域，但它需要一个参考序列来设计引物，这强调了对精确的新质粒组装工具的需求。一些富含gc和重复的区域难以用任何方法解析，这表明在设计新的遗传结构时应优先考虑容易测序的遗传部分。图形抽象
Despite the wide use of plasmids in research and clinical production, the verification of plasmid sequences is a bottleneck that is too often overlooked 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 forde novoassembly. Here, we evaluated short-read, long-read, and hybrid (both short and long reads)de novoassembly pipelines across three replicates of a 24-plasmid library. Consistent with previous characterizations of each sequencing technology, short-read assemblies had frequent 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 consistent assembly generation. Although Sanger sequencing can be used to verify specific regions, it requires a reference sequence to design primers, emphasizing the need for accuratede novoplasmid assembly tools. Some GC-rich and repetitive regions were difficult to resolve using any methods, suggesting that easily sequenced genetic parts should be prioritized in the design of new genetic constructs.GRAPHICAL ABSTRACT