使用基于纳米孔测序的长读长质粒测序与组装工作流程
July 7th, 2026
本研究描述了当染色体序列已知或无兴趣时，质粒纳米孔测序的工作流程。初始质粒DNA富集步骤确保质粒序列深度最优，并最大化每个流细胞可测序的样本数量。
我们研究自我复制的环状DNA元素（质粒）的复制、维持和传播，这需要确定其序列。该方案通过富集质粒DNA和优化组装方法，解决了长读序列测序相关的覆盖率和准确性问题。我们的方案旨在对染色体序列已知或无关情形进行质粒测序。
首先，将样本划线到LB螺旋板上，并补充相应抗生素，以选择相应的质粒携带耐药标记。将平板在37摄氏度下孵育24小时。次日，对单个分离菌落接种3至5毫升LB培养基，辅以相应抗生素。
在37摄氏度下摇晃培养培养约八小时，直到600纳米的光学密度达到一到两小时。然后接种400毫升LB培养基，配合相应抗生素，并与400微升起始培养液接种，并如前所述培养。然后将培养物转移到适合培养体积的离心瓶中。
在4,200克离心机加热10分钟，然后用制造商推荐的试剂体积重新悬挂沉淀。细胞裂解和中和后，将裂解液在4,200克离心30分钟。将上清液转移到新管子中，再次离心。
按照制造商的说明进行平衡、清洗和DNA洗脱。然后将沉淀的DNA重新悬浮在100微升无核酸酶水中，以最大化纳米孔图库制备的浓度。接下来，为了定量DNA，按照所示表格准备检测管。
校准好荧光仪后，将样品管插入荧光仪，并按照仪器手册记录读数。将热循环器编程为30摄氏度两分钟，随后80摄氏度两分钟。按照表格旋转并混合解冻后的套件组件。
利用热循环程序孵育用于条形码的PCR管。然后把管子放冰上冷却。将条形码样本集中在干净的1.5毫升Eppendorf管中，确保总体积不超过1000微升。
珠子涡旋后，向合并的条形样本加入等量的珠子。通过弹动管子混合，然后进行10分钟的孵化。然后用一毫升80%乙醇在磁石上清洗珠子。
去除乙醇，重复一次。把磁铁管子从磁铁上取下来。根据使用条码数量，将珠子重新悬浮在洗脱缓冲液中，并于室温下孵育10分钟。
用一微升样本在荧光仪上定量DNA浓度，以确认文库制备成功。接头连接时，将11微升DNA文库转移到新管子中并进行标记。然后将呈现的成分混合在新管中，稀释解冻后的快速适配器。
接着，将稀释的快速接头1微升加入11微升DNA库中，轻轻拨动管子混合内容物。短暂旋转管子，确保所有体积集中在底部，然后在工作台上孵育五分钟。将流细胞引介混合物置于新鲜的DNA管中，通过倒置和移液混合。
掀开装置盖子，将流孔插入，同时轻轻施加压力以确保正确放置。检查引水口内或附近是否有气泡后，抽取20至30微升液体以去除气泡。然后抽取800微升的引火液，注入引爆口，同时避免气泡，让设备静置五分钟。
将DNA文库与相应体积的图库珠子混合，放入新管中，如此处所示。将200微升的引气混合液注入流速单元引气口。调整样品端口盖以露出端口。
每次滴入75微升DNA文库和珠子，滴入样本端口，让每一滴吸收后再加入下一滴。然后重新盖上样品口盖，关闭引爆口。设备盖合上后，开始测序运行。
根据被复用质粒数量设定运行时间限制，默认为48小时，最长72小时。选择超精确的基准呼叫模式和相应的快速条码套件选项。然后选择原始的FAST5或POD5数据，开始基调。
为了生成用于下游组装的过滤FASTQ文件，使用Filtlong对长读段进行修剪，以去除约5-10%的最低质量读段，同时保留约90%的总良率。根据推荐设置，使用 Autocycler 读取子样本。使用Raven、Flye、Kanu、Miniasm、Myloasm、NECAT、Plassembler及其他精选汇编器组装每个读取子集。
用绷带可视化每个装配图文件。评估连接体的圆形性、组装器间尺寸一致性及嵌合节理证据。移除跨多个组装器缺乏支持的线性连写，保留具有一致大小估计的圆形连写。
当主要质粒圆化时，在共识生成前移除少量无支持的源物。人工策划后进入共识生成。用Autocycler生成共识序列，并搭配精选的组装。
执行剩余的Autocycler命令，生成最终共识质粒序列。最后，用Medaka修剪长读段打磨最终组装，以提高共识准确率。所有接合检测均产生转缀结合，证实供体中存在接合质粒。
组装后的质粒序列尺寸与基于脉冲场凝胶电泳迁移率的估计值一致。纳米孔测序为所有分析样品提供了足够的读数输出和质量。染色体污染率在72.1%至28.0%之间，平均为46.9%。对应变质粒UPEC_271脉冲场凝胶电泳分析显示，其质粒条带约为145千碱基对。
其中一个转偶联体的DNA显示序列与145千碱基完全相同。对应激血液进行脉冲场凝胶电泳分析100913鉴定出两个约为70和110千碱基对的质粒。纳米孔测序确认了两个质粒组装体，分别为71,182碱基对和111,226碱基对。
最重要的考虑因素是染色体序列是否值得关注。如果是这样，标准的全基因组测序方案更合适。我们方案覆盖范围的增加为异质学研究打开了大门，即
在特定种群内的质粒序列变异。
Overview
This article presents a detailed workflow for nanopore sequencing aimed at maximizing plasmid DNA yield and sequencing accuracy. The protocol encompasses plasmid extraction, rapid barcoding, adapter ligation, sequencing, and sequence assembly, and is suitable for a wide range of plasmid sizes and levels of chromosomal contamination. The method is demonstrated using Escherichia coli as a model organism.
Key Study Components
Area of Science
- Molecular biology
- Genomics
- Microbiology
Background
- Plasmid sequencing is essential for genetic studies, recombinant gene expression, and plasmid capture experiments.
- Traditional sequencing methods may be limited by chromosomal contamination and low plasmid yield.
- Nanopore sequencing offers long-read capabilities and real-time data acquisition.
- Efficient workflows are needed to optimize plasmid sequencing throughput and accuracy.
Purpose of Study
- To develop and describe a robust workflow for nanopore-based plasmid sequencing.
- To maximize plasmid DNA yield and sequencing accuracy.
- To enable efficient parallel sequencing of multiple samples.
Methods Used
- Plasmid DNA extraction from clinical E. coli strains.
- Library preparation using rapid barcoding and adapter ligation.
- Loading libraries onto a nanopore flow cell for sequencing.
- Base calling performed in real time or post-hoc.
- Sequence assembly using Autocycler.
- Comparison of assemblies to gel mobility and short-read polished assemblies.
Main Results
- Plasmid assemblies matched sizes inferred from gel mobility.
- In two-thirds of cases, assemblies matched or closely resembled short-read polished assemblies (within 0.05% pairwise identity).
- Protocol supports plasmids from 4 to 174 kb in size.
- Up to 72% chromosomal contamination tolerated without loss of accuracy.
- Parallel sequencing of up to 24 samples achieved with optimal coverage.
Conclusions
- The workflow enables accurate and efficient plasmid sequencing using nanopore technology.
- It is suitable for plasmid verification, improving WGS-derived plasmid sequences, and sequencing plasmids captured by conjugation.
- The protocol is robust to chromosomal contamination and applicable to a range of plasmid sizes.
Frequently Asked Questions
Executive Industry Relevance
Long-read nanopore plasmid sequencing enables high-confidence verification and assembly of plasmid constructs, supporting critical quality control and mechanistic de-risking in biopharma R&D. This workflow addresses the challenge of chromosomal contamination and variable plasmid yield, providing robust, scalable solutions for recombinant DNA and gene therapy vector development. Its parallelization and tolerance for mixed DNA inputs streamline early discovery and preclinical workflows, enhancing portfolio decision-making.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables precise verification of engineered plasmids for functional studies and target validation.
- Reduces ambiguity in construct identity, supporting mechanistic de-risking in discovery biology.
- Facilitates rapid triage of candidate constructs for downstream screening or expression.
Screening & Assay Development
- Provides validated sequence data for assay-ready plasmids, ensuring reproducibility in screening campaigns.
- Supports parallel sequencing of up to 24 samples, increasing throughput for construct libraries.
- Delivers quantitative sequence accuracy, enabling reliable comparison across engineered variants.
Translational & Preclinical Research
- Improves continuity from discovery to preclinical studies by confirming plasmid integrity in disease-relevant systems.
- Enables sequence verification for plasmids used in gene delivery or biomarker studies.
- Supports risk-adjusted advancement by minimizing sequence-related uncertainties in preclinical models.
Pipeline & Workflow Integration
This nanopore-based workflow integrates from early construct verification through screening and preclinical validation, supporting both hypothesis-driven and translational research pipelines.
- Discovery Biology: Confirms plasmid sequence fidelity, reducing risk of off-target effects in functional assays.
- Screening: Ensures assay-ready constructs are sequence-verified, supporting reproducible compound evaluation.
- Analytics: Provides high-accuracy, quantitative sequence data for cross-sample comparison and quality control.
- Translational Research: Aligns plasmid sequence verification with preclinical model requirements for gene expression studies.
- Enterprise Reuse: Offers a scalable, parallelizable sequencing capability adaptable across diverse R&D programs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in construct-driven studies and reduces mechanistic ambiguity.
- Operational Value: Standardizes plasmid verification, tolerates high chromosomal contamination, and supports high-throughput workflows.
- Strategic Value: Enables better go/no-go decisions by ensuring construct integrity before downstream investment.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of gene-based assets.
Implementation Considerations
- Requires expertise in plasmid extraction, library preparation, and nanopore sequencing workflows.
- Demands computational infrastructure scaled to real-time or post-hoc basecalling needs.
- Benefits from standardized protocols for cross-team reproducibility and data comparability.
- Adaptable to a range of plasmid sizes and host backgrounds, including high chromosomal DNA content.
- Best suited for applications where chromosomal sequence is known or not required.
