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J Clin Microbiol. 2017 Feb;55(2):606-615. doi: 10.1128/JCM.02121-16. Epub 2016 Dec 7.

High-Throughput Next-Generation Sequencing of Polioviruses.

Author information

1
CSRA International, Atlanta, Georgia, USA.
2
Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA ylz9@cdc.gov.
3
IHRC, Atlanta, Georgia, USA.
4
Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
5
Oak Ridge Institute for Science and Education, Oak Ridge, Tennessee, USA.
6
Centre Pasteur du Cameroon, Yaoundé, Cameroon.
7
Department of Virology, College of Medicine, University of Ibadan, Ibadan, Nigeria.

Abstract

The poliovirus (PV) is currently targeted for worldwide eradication and containment. Sanger-based sequencing of the viral protein 1 (VP1) capsid region is currently the standard method for PV surveillance. However, the whole-genome sequence is sometimes needed for higher resolution global surveillance. In this study, we optimized whole-genome sequencing protocols for poliovirus isolates and FTA cards using next-generation sequencing (NGS), aiming for high sequence coverage, efficiency, and throughput. We found that DNase treatment of poliovirus RNA followed by random reverse transcription (RT), amplification, and the use of the Nextera XT DNA library preparation kit produced significantly better results than other preparations. The average viral reads per total reads, a measurement of efficiency, was as high as 84.2% ± 15.6%. PV genomes covering >99 to 100% of the reference length were obtained and validated with Sanger sequencing. A total of 52 PV genomes were generated, multiplexing as many as 64 samples in a single Illumina MiSeq run. This high-throughput, sequence-independent NGS approach facilitated the detection of a diverse range of PVs, especially for those in vaccine-derived polioviruses (VDPV), circulating VDPV, or immunodeficiency-related VDPV. In contrast to results from previous studies on other viruses, our results showed that filtration and nuclease treatment did not discernibly increase the sequencing efficiency of PV isolates. However, DNase treatment after nucleic acid extraction to remove host DNA significantly improved the sequencing results. This NGS method has been successfully implemented to generate PV genomes for molecular epidemiology of the most recent PV isolates. Additionally, the ability to obtain full PV genomes from FTA cards will aid in facilitating global poliovirus surveillance.

KEYWORDS:

FTA cards; cell culture; metagenomics; next-generation sequencing; picornavirus; poliovirus

PMID:
27927929
PMCID:
PMC5277531
DOI:
10.1128/JCM.02121-16
[Indexed for MEDLINE]
Free PMC Article

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