Real Time Nanopore Sequencing at Scale - Read Until and Run Until on PromethION scale devices.

大规模实时纳米孔测序 - 在 PromethION 规模设备上“Read Until”和“Run Until”。

• 批准号: 2432086
• 金额: --
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2432086
• 关键词: Real; Time; Nanopore; Sequencing; Scale

Rapid sequencing of nucleic acids transformed our understanding of the biological world. New technologies and methods provide new challenges developing optimal approaches to address biological questions. Only Oxford Nanopore Technologies (ONT) instruments enable real time sequencing where sequence data can be analysed during data generation. Nanopore sequencing works by measuring current flow as DNA passes through a small nanopore. Current flow depends on the sequence of DNA present and so generates a current trace which can be decoded revealing the DNA sequence.We have exploited this technology in a number of ways, notably establishing "read until". Here the start of a molecule is analysed whilst being sequenced. If the molecule matches a region of interest, sequencing can continue. If not the voltage over the specific channel can be reversed and the read rejected. In principle, this enables selective sequencing of specific molecules from a library. We were the first to demonstrate that this method works using dynamic time warping to map the nanopore squiggle to a reference. Subsequently, working through an iCASE studentship between ONT and Nottingham, we have jointly developed an approach to basecall sequence data directly from the squiggle trace whilst sequencing. This method removes limitations of searching within signals and can be readily used by others with access to the same hardware. At present, the method is limited to a specific subtype of ONT sequencers (the GridION and MinION). These platforms have the capacity to generate 10-20 Gb of data per flowcell. The PromethION sequencer can generate in excess of 100 Gb of sequence data per flowcell from up to 48 positions simultaneously. This enables sequencing of a human genome within 60 hours on a single flowcell with long reads. In principle, selective sequencing at this scale could enable many useful applications such as a low coverage whole genome coupled with higher coverage of specific regions such as known cancer-causing SNPs or other structural variants of interest.However, "read until" has a negative impact on the flowcell performance (particularly on the PromethION) and so experimental design requires optimisation depending on the precise experimental goal. We are working with ONT to determine the underlying reasons for this and develop methods to implement "Read Until" on higher throughput platforms. In this studentship we will develop methods to implement two real time approaches around nanopore sequencing. 1) Run Until, whereby sequencing can be automatically halted once a specific experimental goal has been achieved and, 2), coupling this iterative type of approach with 'read until' such that once a specific goal has been achieved, reads can be rejected which are no longer required. Methods will include genome assembly, targeted coverage of regions of whole genomes, and filtering of metagenomes. We will also look to establish methods that can exploit one or more sequencing positions simultaneously or sequentially. Crucially, these methods must be dynamic and offer flexibility over and above custom library preparation which might enrich for specific targets.

核酸的快速测序改变了我们对生物世界的理解。新的技术和方法带来了开发解决生物学问题的最佳方法的新挑战。只有牛津纳米孔技术(ONT)仪器能够实现实时测序，其中序列数据可以在数据生成期间进行分析。纳米孔测序的工作原理是测量DNA通过小纳米孔时的电流流量。电流取决于存在的DNA序列，因此产生的电流痕迹可以被解码，揭示DNA序列。我们已经通过多种方式利用了这项技术，特别是建立了“读到”。这里对分子的起始进行了分析，同时对其进行了测序。如果分子与感兴趣的区域匹配，测序就可以继续。如果不是，则可以反转特定通道上的电压并且拒绝读取。原则上，这可以对文库中的特定分子进行选择性测序。我们第一个演示了这种方法的工作原理，它使用动态时间扭曲将纳米孔弯曲映射到参考。随后，通过ONT和诺丁汉之间的iCASE学生合作，我们共同开发了一种在测序的同时直接从弯曲轨迹获得基本序列数据的方法。这种方法消除了在信号内搜索的限制，并且可以很容易地被访问相同硬件的其他人使用。目前，该方法仅限于ONT定序器的特定子类型(GridION和Minion)。这些平台具有每个Flow cell产生10-20 GB数据的能力。Prometion测序仪可以同时从多达48个位置为每个流动细胞生成超过100 GB的序列数据。这使得能够在60小时内通过长时间读取的单个流动细胞对人类基因组进行测序。原则上，这种规模的选择性测序可以实现许多有用的应用，例如低覆盖率的全基因组和更高覆盖率的特定区域，如已知的致癌SNPs或其他感兴趣的结构变体。然而，阅读直到对流式细胞的性能(特别是对繁殖)有负面影响，因此实验设计需要根据确切的实验目标进行优化。我们正在与ONT合作，确定造成这种情况的根本原因，并开发方法，在更高吞吐量的平台上实现“读到”。在这次学习中，我们将开发两种方法来实现纳米孔测序的两种实时方法。1)运行到，其中一旦已经达到特定的实验目标，就可以自动停止测序，以及，2)将这种迭代类型的方法与‘读取直到’相结合，使得一旦已经实现了特定的目标，就可以拒绝不再需要的读取。方法将包括基因组组装，有针对性地覆盖整个基因组的区域，以及筛选元基因组。我们还将寻求建立能够同时或顺序利用一个或多个测序位置的方法。至关重要的是，这些方法必须是动态的，并且在定制库准备的基础上提供灵活性，这可能会丰富特定的目标。