Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples
Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples
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DOI：
10.1038/nprot.2017.066
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发表时间：
2017-06-01
期刊：
影响因子：
14.8
通讯作者：
Loman, Nicholas J.
中科院分区：
文献类型：
--
作者：
Quick, Joshua;Grubaugh, Nathan D.;Loman, Nicholas J.
基因组测序已成为研究新出现的传染病的有力工具；然而，直接从临床样本中进行基因组测序(即，无需分离和培养)对寨卡病毒等病毒仍然具有挑战性，对于这些病毒，元基因组测序方法可能产生不足的病毒读数。在这里，我们提出了一种生成编码序列完整基因组的方法，包括一个在线引物设计工具，一个新的多重PCR富集法，为便携式Minion测序仪(Oxford Nanopore Technologies)和Illumina系列仪器优化的文库制备方法，以及用于生成一致序列的生物信息学流水线。Minion协议不需要互联网连接即可进行分析，因此适用于连接有限的现场应用程序。我们的方法依赖于多重聚合酶链式反应，从每次反应只有50个基因组拷贝的样本中进行有针对性的病毒基因组浓缩。通过从临床样本开始并遵循简单的实验室工作流程，可以在1-2天内实现病毒共识序列。这种方法已经被几个研究寨卡病毒进化的小组成功使用，并正在促进对病毒在美洲传播的了解。该协议可用于使用在线Primal Program Primer Designer软件对其他病毒基因组进行测序。它适合于在暴发期间在现场对RNA或DNA病毒进行测序，或者作为一种廉价、方便的实验室使用方法。
Genome sequencing has become a powerful tool for studying emerging infectious diseases; however, genome sequencing directly from clinical samples (i.e., without isolation and culture) remains challenging for viruses such as Zika, for which metagenomic sequencing methods may generate insufficient numbers of viral reads. Here we present a protocol for generating coding-sequence-complete genomes, comprising an online primer design tool, a novel multiplex PCR enrichment protocol, optimized library preparation methods for the portable MinION sequencer (Oxford Nanopore Technologies) and the Illumina range of instruments, and a bioinformatics pipeline for generating consensus sequences. The MinION protocol does not require an Internet connection for analysis, making it suitable for field applications with limited connectivity. Our method relies on multiplex PCR for targeted enrichment of viral genomes from samples containing as few as 50 genome copies per reaction. Viral consensus sequences can be achieved in 1-2 d by starting with clinical samples and following a simple laboratory workflow. This method has been successfully used by several groups studying Zika virus evolution and is facilitating an understanding of the spread of the virus in the Americas. The protocol can be used to sequence other viral genomes using the online Primal Scheme primer designer software. It is suitable for sequencing either RNA or DNA viruses in the field during outbreaks or as an inexpensive, convenient method for use in the lab.