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

优化纳米孔测序技术中的自适应采样（“Read Until”）方法

• 批准号: BB/N018877/1
• 负责人: Guy Cochrane
• 金额: $ 39.59万
• 依托单位: European Bioinformatics Institute
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FN018877%2F1
• 关键词: Adaptive; sampling; Read; Until; methods

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.

在过去的三十年里，DNA测序已经成为跨越生命科学甚至超越生命科学的一项关键技术。事实上，生物学研究的几乎所有领域都被直接使用的技术或从别人的工作中获得的知识所触及，在这些工作中已经使用了测序。这项技术发展迅速。在2000年代中期，第二代测序技术与第一代完全不同，它带来了测序机运行速度的阶段性变化，并相应地大幅降低了成本。这些技术现在占据主导地位，并带来了大量新的和有影响力的科学发现，尤其是作为数千个动物、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波技术浪潮的风口浪尖，也就是纳米孔测序，这与之前的技术完全不同，它有望实现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，并将重点放在该技术中一个特定的、尚未被探索的、有望产生非常重大影响的特征上。纳米孔测序使用可以工程设计和组织到表面上的微孔。这些小孔允许DNA分子一次一个地从表面的一边到另一边通过。当它们通过时，孔提供通过孔内表面的碱基(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)放在小孔上方，然后小孔捕获DNA分子的末端，并开始将其吸过，同时读取其序列。该系统的控制是如此精细，如果需要的话，DNA分子可以在完全测序之前被排除在孔中，捕获过程可以迅速重新开始。所有测序平台面临的一个关键挑战是，基因组的某些部分很难测序，而其他部分则不是。正因为如此，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(通常是30次)读取基因组，以便至少读取一次困难的区域。通过自适应采样，我们计划用软件克服这一障碍，该软件将快速从毛孔中读取早期序列，并决定从毛孔中出现的基因组部分是否已经被读取。根据这一点，可以决定是将DNA分子从毛孔中排斥出来，还是将阅读进行到底。通过避免以这种方式重新测序将节省大量时间，从而推动更具成本效益、更快速和更有针对性的测序。虽然我们的技术将广泛使用，但我们将专门针对五个示例挑战进行工作，在这些挑战中，工具将是有用的。这些工作包括检测和鉴定传染病细菌，研究农业牲畜，调查农作物基因组，研究养殖鱼类以了解对致病物种的反应，以及分析环境中微生物物种的群落。这种方法有很大的新颖性。在之前有关埃博拉病毒的工作中，我们已经表明，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，在这种方法中，数据分析(以前在测序完成后发生的事情)直接影响到物理测序机本身在测序实验期间的操作方式。作为该项目的一部分，我们计划发布软件并举办两次研讨会，向技术员、基因组实验室、研究科学家和工业界传播我们开发的产品。

项目成果

The European Nucleotide Archive in 2020.

• DOI: 10.1093/nar/gkaa1028
• 发表时间: 2021-01-08
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Harrison PW;Ahamed A;Aslam R;Alako BTF;Burgin J;Buso N;Courtot M;Fan J;Gupta D;Haseeb M;Holt S;Ibrahim T;Ivanov E;Jayathilaka S;Balavenkataraman Kadhirvelu V;Kumar M;Lopez R;Kay S;Leinonen R;Liu X;O'Cathail C;Pakseresht A;Park Y;Pesant S;Rahman N;Rajan J;Sokolov A;Vijayaraja S;Waheed Z;Zyoud A;Burdett T;Cochrane G
• 通讯作者: Cochrane G

Nanopore sequencing and assembly of a human genome with ultra-long reads.

• DOI: 10.1038/nbt.4060
• 发表时间: 2018-04
• 期刊: Nature biotechnology
• 影响因子: 46.9
• 作者: Jain M;Koren S;Miga KH;Quick J;Rand AC;Sasani TA;Tyson JR;Beggs AD;Dilthey AT;Fiddes IT;Malla S;Marriott H;Nieto T;O'Grady J;Olsen HE;Pedersen BS;Rhie A;Richardson H;Quinlan AR;Snutch TP;Tee L;Paten B;Phillippy AM;Simpson JT;Loman NJ;Loose M
• 通讯作者: Loose M

Dynamic, adaptive sampling during nanopore sequencing using Bayesian experimental design

使用贝叶斯实验设计在纳米孔测序过程中动态、自适应采样

• DOI: 10.1101/2020.02.07.938670
• 发表时间: 2020
• 作者: Weilguny L

Dynamic, adaptive sampling during nanopore sequencing using Bayesian experimental design.

• DOI: 10.1038/s41587-022-01580-z
• 发表时间: 2023-07
• 期刊: NATURE BIOTECHNOLOGY
• 影响因子: 46.9
• 作者: Weilguny, Lukas;De Maio, Nicola;Munro, Rory;Manser, Charlotte;Birney, Ewan;Loose, Matthew;Goldman, Nick
• 通讯作者: Goldman, Nick