Science及Nature共述:口袋里的DNA测序仪,可实现实时诊断
2016/02/23
2016年2月3日,《Nature》阐述了纳米孔测序现场检测埃博拉病毒的成功例子, 2016年2月19日,《Science》发文与《Nature》共同阐述了纳米孔测序的未来。


纳米孔测序的想法起始于25年前, 2012年5月4日《Science》首次报道了Oxford Nanopore公司研制的纳米孔测序仪样机——MinION,用于破译病毒DNA。然而MinION的发展历程并不那么顺利,英国牛津大学基因组学中心基因组学家Rory Bowden说,“众所周知,在每次读取的水平上,MinION不是很精确”。2014年2月《Science》表示为了正确读取每个碱基,纳米孔的数据必须结合常规测序数据进行分析。2016年2月3日,《Nature》阐述了纳米孔测序现场检测埃博拉病毒的成功例子, 2016年2月19日,《Science》以《Pocket DNA sequencers make real-time diagnostics a reality》为题,再次阐述了纳米孔测序的未来。

MinION一直在探索的路上

到目前为止,大多数的测序是通过构建待测链的互补链而实现,同时必须用化学标记碱基,以方便确定它们是否被逐一加到新链中,此外这种技术将产生许多需要拼接的小片段。纳米孔的方法可更直接地阅读碱基,当单链DNA通过微孔时,每个碱基以独有的方式中断孔隙中的离子流以揭示其身份。在纳米孔测序之前,基因测序需要大量的设备、时间及金钱。而纳米孔测序的问世,使“行李箱中的基因测序成为了可能”,然而,迄今为止只有一家公司生产此类测序仪,且准确率比较低下。

在过去两年里,数以百计的实验室在尝试MinION。例如,上个月新加坡基因组研究所计算机生物学家Niranjan Nagarajan领导的团队报道了一种无需修改测序程序便可提高测序精确度的方法。该研究团队利用MinION来确定皮肤或粪便样本中的细菌。为了区分细菌物种,研究人员测定了每个样本的16s核糖体基因序列。传统的测序方法只能检测出基因的一部分,有时不足以进行阳性分析。MinION可捕获更多甚至是全部的基因信息,这使得物种的鉴定更加精确——若序列足够精确。

为了提高准确性,Nagarajan利用化学物质将16S基因制成环形,并添加特殊的DNA复制酶对环形DNA进行复制,从而产生多个重复的DNA片段。当每个字符串经过MinION孔时,16S基因便被多次测序,大约重复6次测序足以保证精准识别每个碱基。研究人员于1月27日在《bioRxiv》公布了该结果。

《Nature》和《Science》共述,MinION可实现实时诊断


2016年2月3日,《Nature》首次报道了利用纳米孔测序对埃博拉患者样本进行实时测序的成功例子,同时2月19日《Science》也对此事件进行了阐述,《Science》表示埃博拉现场测序的成功取决于MinION精准度的提高,在现场检测埃博拉病毒序列的同时,其他研究人员在实验室中调整样品制备和数据分析以提高设备的精度和速度。

英国伯明翰大学微生物基因组学家Nicholas Loman及其同事意识到可从碱基通过孔隙时离子流的变化进一步提取碱基的信息。巴尔的摩约翰霍普金斯大学生物医学工程师Winston Timp说,电流信号中蕴藏着更多的信息。每个碱基信号都受其两侧周围的影响。联合使用分析“波形曲线”的新的计算机程序(该程序由多伦多安大略癌症研究所 Jared Simpson等人研发),该研究团队决定单独使用纳米孔数据来分析细菌序列。

随后研究人员将他们的序列带到西非,在那里他们成功地从患者身上检测出了148株埃博拉病毒基因组。即使在田间条件下,研究人员也可在24小时内完成一个基因组测序,研究人员表示,实时分析病原体将触手可及。

生态学家、公共卫生官员、流行病理学家、食品安全官员以及其他人员都将受益于此。参与研发该测序仪的加州大学分子生物学家Mark Akeson说,“纳米孔测序的出发点是在星球上进行DNA测序,是一种民主化测序。”

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    Not so long ago, DNA sequencing required massive equipment and lots of time and money. Now, relatively cheap, pocket-sized devices are on the verge of giving real-time sequencing abilities to the masses. These so-called nanopore sequencers, produced so far by a single company, have suffered from poor accuracy. But this month, researchers reported that the instruments passed an important field test, conducting on-the-spot sequencing of viruses isolated from patients during last year's Ebola epidemic in West Africa. In the lab, meanwhile, other researchers are tweaking sample preparation and data analysis to boost the devices' accuracy and speed. Real-time analyses of pathogens and the rest of life are within reach, they say. Ecologists, public health officials, epidemiologists, food safety officials, and many others may reap the benefits. Some researchers predict that one day these sequencers will be in every lab and even in everyone's pocket, like mobile phones.

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  • Real-time, portable genome sequencing for Ebola surveillance

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