Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples
By
Josh Quick,
Nathan D. Grubaugh,
Steven T Pullan,
Ingra M Claro,
Andrew D Smith,
Karthik Gangavarapu,
Glenn Oliveira,
Refugio Robles-Sikisaka,
Thomas F Rogers,
Nathan A Beutler,
Dennis R. Burton,
Lia Laura Lewis-Ximenez,
Jaqueline Goes de Jesus,
Marta Giovanetti,
Sarah Hill,
Allison Black,
Trevor Bedford,
Miles W Carroll,
Marcio Nunes,
Luiz Carlos Alcantara,
Ester C Sabino,
Sally A. Baylis,
Nuno Faria,
Matthew Loose,
Jared T Simpson,
Oliver G Pybus,
Kristian G. Andersen,
Nick J Loman
Posted 09 Jan 2017
bioRxiv DOI: 10.1101/098913
(published DOI: 10.1038/nprot.2017.066)
Genome sequencing has become a powerful tool for studying emerging infectious diseases; however, genome sequencing directly from clinical samples without isolation remains challenging for viruses such as Zika, where 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, optimised 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 starting with clinical samples in 1-2 days 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.
Download data
- Downloaded 4,668 times
- Download rankings, all-time:
- Site-wide: 2,048
- In genomics: 219
- Year to date:
- Site-wide: 21,129
- Since beginning of last month:
- Site-wide: 17,434
Altmetric data
Downloads over time
Distribution of downloads per paper, site-wide
PanLingua
News
- 27 Nov 2020: The website and API now include results pulled from medRxiv as well as bioRxiv.
- 18 Dec 2019: We're pleased to announce PanLingua, a new tool that enables you to search for machine-translated bioRxiv preprints using more than 100 different languages.
- 21 May 2019: PLOS Biology has published a community page about Rxivist.org and its design.
- 10 May 2019: The paper analyzing the Rxivist dataset has been published at eLife.
- 1 Mar 2019: We now have summary statistics about bioRxiv downloads and submissions.
- 8 Feb 2019: Data from Altmetric is now available on the Rxivist details page for every preprint. Look for the "donut" under the download metrics.
- 30 Jan 2019: preLights has featured the Rxivist preprint and written about our findings.
- 22 Jan 2019: Nature just published an article about Rxivist and our data.
- 13 Jan 2019: The Rxivist preprint is live!