Adaptive sampling ('Read Until') methods in optimised nanopore sequencing technologies

在过去的三十年里，DNA测序已经成为跨越生命科学的关键技术。事实上，生物学研究中很少有领域没有受到直接使用技术或知识的影响，这些技术或知识来自于使用测序的其他工作。技术进步很快。在2000年代中期，第二代测序技术与第一代测序技术完全不同，它使测序机器的运行速度发生了飞跃性的变化，并相应地大幅降低了成本。这些技术现在占主导地位，并导致了大量新的和有影响力的科学发现，尤其是作为成千上万的动物，植物，真菌和细菌项目背后的核心测序技术。我们现在正处于第三次技术浪潮的风口浪尖，“纳米孔”测序，再次与那些进行它的人完全不同，它承诺类似的改变游戏规则的进步。在“自适应采样”项目中，我们认识到纳米孔测序的潜力，并专注于这项技术的一个特殊功能，但尚未得到充分开发，该技术有望产生非常重大的影响。纳米孔测序使用可以设计和组织到表面上的微观孔。这些孔允许DNA分子一次一个地从表面的一侧通过到另一侧。当它们通过时，孔提供通过孔的内表面的碱基（A、C、G和T）的直接读出。使用者将DNA分子的混合物（整个基因组的片段）放在小孔上，小孔会抓住DNA分子的末端，并开始将其拉过，同时阅读其序列。该系统的控制非常精细，如果需要，可以在完全测序之前将DNA分子从孔中排除，并快速重新开始捕获过程。所有测序平台的一个关键挑战是基因组的某些部分“难以测序”，而其他部分则不然。因此，为了确保基因组测序实验已经捕获了基因组的所有部分，用户必须将实验设置为多次读取基因组（通常为30次），以便至少读取一次困难的区域。有了自适应采样，我们计划用软件来克服这个障碍，该软件将从孔中快速读取早期序列，并决定从孔中出现的基因组部分是否已经被读取或尚未被读取。基于此，可以决定是否将DNA分子从孔中排除或进行阅读直到结束。通过避免以这种方式重新测序所节省的时间将是巨大的，从而推动更经济、快速和“有针对性”的测序。虽然我们的技术将广泛适用，但我们将具体针对五个示例挑战，其中工具将是有用的。这些项目包括检测和鉴定传染性细菌、研究农业牲畜、调查作物基因组、研究养殖鱼类以了解对致病物种的反应以及分析环境中的微生物物种群落。这一方法具有很大的新奇。在之前关于埃博拉病毒的工作中，我们已经证明，使用我们的软件原型拒绝读取具有潜力。据我们所知，我们现在提出的将是第一个测序方法的例子，其中数据分析（以前是在测序完成后发生的事情）对测序实验期间物理测序机本身的操作方式有直接影响。作为该项目的一部分，旨在为研究界带来尽可能广泛的利益，我们计划出版该软件，并举办两次讲习班，向技术专家、基因组学实验室、研究科学家和工业界传播我们开发的软件。

Over the last three decades, DNA sequencing has become a key technology across and beyond the life sciences. Indeed, few areas of biological research remain untouched by either the direct use of the technology or knowledge that is derived from others' work in which sequencing has been used. The technology has advanced rapidly. In the mid-2000s, a second generation of sequencing technologies, quite unlike the first, brought a step change in the rate at which sequencing machines could operate, and a corresponding vast reduction in the cost. These technologies now dominate and have led to a wealth of new and impactful scientific findings, not least as the core sequencing technology behind many thousands of animal, plant, fungal and bacterial projects. We are now on the cusp of a third-wave of technology, 'nanopore' sequencing, again quite unlike those that proceed it, that promises similar game-changing advances. In the 'Adaptive Sampling' project, we recognise the potential of nanopore sequencing and focus on a particular, as yet under-explored, feature of the technology that promises very significant impact.Nanopore sequencing uses microscopic pores that can be engineered and organised onto a surface. The pores allow DNA molecules to pass through one at a time from one side of the surface to the other. As they transit, the pores provide a direct read-out of the bases (A, C, G and T) that pass the inner surface of the pore. The user places a mixture of DNA molecules (fragments of a whole genome) above the pore, which then captures the end of a DNA molecule and starts to draw it through, reading its sequence as it goes. The control of the system is so refined that, if desired, a DNA molecule can be rejected from a pore before it has been fully sequenced and the capture process can start again rapidly.A key challenge for all sequencing platforms is that some parts of genomes are 'difficult to sequence' and others are not. Because of this, to be certain that a genome sequencing experiment has captured all parts of a genome, the user must set the experiment up to read the genome many times (often 30), so that the difficult regions are read at least once. With Adaptive Sampling, we plan to overcome this obstacle with software that will rapidly read the early sequence from a pore, and make a decision about whether the part of the genome that is emerging from the pore has been read already or is yet to be read. Based on this, a decision can be made as to whether or not to reject the DNA molecule from the pore or to carry on reading to the end. The time saving to be achieved by avoiding re-sequencing in this way will be substantial, driving at far more cost-effective, rapid and 'targeted' sequencing.While our technology will be useful broadly, we will work specifically with five example challenges, in which the tools will be useful. These cover detection and identification of infectious bacteria, the study of agricultural livestock, investigation of crop plant genomes, work on farmed fish to understand responses to disease-causing species and the analysis of communities of microbial species in the environment. There is substantial novelty in this approach. In previous work on Ebola virus, we have shown that rejecting reads using a prototype of our software has potential. What we now propose will be the first example, to the best of our knowledge, of a sequencing approach in which data analysis (previously something that happened after sequencing was completed) has direct impact on the way in which the physical sequencing machine itself is operated during a sequencing experiment.As part of the project, aiming at the broadest possible benefit to the research community, we plan to publish the software and hold two workshops in which we disseminate what we have developed to technologists, genomics laboratories, research scientists and industry.