Journal article
Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples
- Abstract:
- 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.
- Publication status:
- Published
- Peer review status:
- Peer reviewed
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- Files:
-
-
(Preview, Accepted manuscript, pdf, 667.9KB, Terms of use)
-
- Publisher copy:
- 10.1038/nprot.2017.066
Authors
+ National Institute of Allergy and Infectious Diseases
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- Funding agency for:
- Andersen, KG
- Grant:
- HHSN272201400048C
+ Sir Henry Dale Fellowship
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- Funding agency for:
- Faria, NR
- Grant:
- 204311/Z/16/Z
+ National Institutes of Health
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- Funding agency for:
- Grubaugh, ND
- Black, A
- Bedford, T
- Andersen, KG
- Grant:
- 5T32AI007244-33
- DGE-1256082
- GM111274
- HHSN272201400048C
+ USAID Emerging Pandemic Threats Program-2 PREDICT-2
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- Grant:
- AID-OAA-A-14-00102
- Publisher:
- Nature Publishing Group
- Journal:
- Nature Protocols More from this journal
- Volume:
- 12
- Issue:
- 4
- Pages:
- 1261–1276
- Publication date:
- 2017-05-24
- DOI:
- EISSN:
-
1750-2799
- ISSN:
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1754-2189
- Language:
-
English
- Keywords:
- Pubs id:
-
pubs:697616
- UUID:
-
uuid:7f72bb6c-9d7f-4ede-8403-ce8c4e03308d
- Local pid:
-
pubs:697616
- Source identifiers:
-
697616
- Deposit date:
-
2017-06-22
- ARK identifier:
Terms of use
- Copyright holder:
- © Quick, et al 2017
- Copyright date:
- 2017
- Notes:
- This is the author accepted manuscript following peer review version of the article. The final version is available online from Nature Publishing Group at: 10.1038/nprot.2017.066
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