Recombinant plasmid vectors are versatile tools that have facilitated discoveries in molecular biology, genetics, proteomics, and many other fields. As the enzymatic and bacterial processes used to create recombinant DNA can introduce errors, sequence validation is an essential step in plasmid assembly. Sanger sequencing is the current standard for plasmid validation; however, this method is limited by an inability to sequence through complex secondary structure and lacks scalability when applied to full-plasmid sequencing of multiple plasmids owing to read-length limits. Although high-throughput sequencing does provide full-plasmid sequencing at scale, it is impractical and costly when used outside of library-scale validation. Here, we present Oxford nanopore-based rapid analysis of multiplexed plasmids (OnRamp), an alternative method for routine plasmid validation that combines the advantages of high-throughput sequencing's full-plasmid coverage and scalability with Sanger's affordability and accessibility by leveraging nanopore's long-read sequencing technology. We include customized wet-laboratory protocols for plasmid preparation along with a pipeline designed for analysis of read data obtained using these protocols. This analysis pipeline is deployed on the OnRamp web app, which generates alignments between actual and predicted plasmid sequences, quality scores, and read-level views. OnRamp is designed to be broadly accessible regardless of programming experience to facilitate more widespread adoption of long-read sequencing for routine plasmid validation. Here we describe the OnRamp protocols and pipeline and show our ability to obtain full sequences from pooled plasmids while detecting sequence variation even in regions of high secondary structure at less than half the cost of equivalent Sanger sequencing.

中文翻译：

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使用 OnRamp 进行多重长读长质粒验证和分析


重组质粒载体是一种多功能工具，促进了分子生物学、遗传学、蛋白质组学和许多其他领域的发现。由于用于创建重组 DNA 的酶促和细菌过程可能会引入错误，因此序列验证是质粒组装中的重要步骤。桑格测序是当前质粒验证的标准；然而，该方法由于无法通过复杂的二级结构进行测序而受到限制，并且由于读长限制，当应用于多个质粒的全质粒测序时缺乏可扩展性。尽管高通量测序确实提供了大规模的全质粒测序，但在文库规模验证之外使用时，它是不切实际且成本高昂的。在这里，我们提出了基于牛津纳米孔的多重质粒快速分析（OnRamp），这是一种常规质粒验证的替代方法，通过利用纳米孔的长读长，将高通量测序的全质粒覆盖和可扩展性的优势与桑格的经济性和可及性结合起来测序技术。我们包括用于质粒制备的定制湿实验室方案以及设计用于分析使用这些方案获得的读取数据的管道。该分析管道部署在 OnRamp Web 应用程序上，该应用程序生成实际和预测的质粒序列、质量分数和读取级别视图之间的比对。 OnRamp 的设计宗旨是无论编程经验如何，均可广泛访问，以促进更广泛地采用长读长测序进行常规质粒验证。 在这里，我们描述了 OnRamp 协议和流程，并展示了我们从汇集的质粒中获取完整序列的能力，同时检测序列变异，甚至在高二级结构区域中，成本不到同等桑格测序的一半。