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

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新的挑战，需要制定解决生物问题的最佳办法。只有Oxford Nanopore Technologies（ONT）仪器能够实现真实的实时测序，其中可以在数据生成过程中分析序列数据。纳米孔测序通过测量DNA通过小纳米孔时的电流来工作。电流的流动取决于DNA的序列，因此产生一个电流轨迹，可以通过解码揭示DNA序列。我们已经以多种方式利用这项技术，特别是建立“读到”。在这里，在测序的同时分析分子的起始。如果分子匹配感兴趣的区域，则测序可以继续。如果不是，则特定通道上的电压可以被反转并且读取被拒绝。原则上，这使得能够对来自文库的特定分子进行选择性测序。我们是第一个证明这种方法使用动态时间弯曲将纳米孔曲线映射到参考的方法。随后，通过ONT和诺丁汉之间的iCASE学生奖学金，我们共同开发了一种方法，直接从波形图中提取测序数据。这种方法消除了在信号内搜索的限制，并且可以容易地由访问相同硬件的其他人使用。目前，该方法仅限于ONT测序仪的特定亚型（GridION和MinION）。这些平台具有每个流通池生成10-20 Gb数据的能力。PromethION测序仪可以同时从多达48个位置生成每个流通池超过100 Gb的序列数据。这使得人类基因组的测序在60小时内在一个单一的流动池与长的读取。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，加上特定区域（例如已知致癌SNP或其他感兴趣的结构变体）的较高覆盖率。然而，“读到”对流通池性能有负面影响（特别是在PromethION上），因此实验设计需要根据精确的实验目标进行优化。我们正在与ONT合作，以确定这一问题的根本原因，并开发在更高吞吐量平台上实现“Read Until”的方法。在这个学生项目中，我们将开发方法来实现两个围绕纳米孔测序的真实的时间方法。1)Run Until，从而一旦实现了特定的实验目标，测序就可以自动停止，以及2）将这种迭代类型的方法与“read until”结合，使得一旦实现了特定的目标，就可以拒绝不再需要的读数。方法将包括基因组组装、全基因组区域的靶向覆盖和宏基因组的过滤。我们还将寻求建立可以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，并提供超过定制库制备的灵活性，这可能会富集特定的目标。

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.