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Open Access 01-12-2016 | Methodology

Development and evaluation of a non-ribosomal random PCR and next-generation sequencing based assay for detection and sequencing of hand, foot and mouth disease pathogens

Authors: Anh To Nguyen, Thanh Tan Tran, Van Minh Tu Hoang, Ngoc My Nghiem, Nhu Nguyen Truc Le, Thanh Thi My Le, Qui Tu Phan, Khanh Huu Truong, Nhan Nguyen Thanh Le, Viet Lu Ho, Viet Chau Do, Tuan Manh Ha, Hung Thanh Nguyen, Chau Van Vinh Nguyen, Guy Thwaites, H. Rogier van Doorn, Tan Van Le

Published in: Virology Journal | Issue 1/2016

Abstract

Background

Hand, foot and mouth disease (HFMD) has become a major public health problem across the Asia-Pacific region, and is commonly caused by enterovirus A71 (EV-A71) and coxsackievirus A6 (CV-A6), CV-A10 and CV-A16. Generating pathogen whole-genome sequences is essential for understanding their evolutionary biology. The frequent replacements among EV serotypes and a limited numbers of available whole-genome sequences hinder the development of overlapping PCRs for whole-genome sequencing.

We developed and evaluated a non-ribosomal random PCR (rPCR) and next-generation sequencing based assay for sequence-independent whole-genome amplification and sequencing of HFMD pathogens. A total of 16 EV-A71/CV-A6/CV-A10/CV-A16 PCR positive rectal/throat swabs (Cp values: 20.9–33.3) were used for assay evaluation.

Results

Our assay evidently outperformed the conventional rPCR in terms of the total number of EV-A71 reads and the percentage of EV-A71 reads: 2.6 % (1275/50,000 reads) vs. 0.1 % (31/50,000) and 6 % (3008/50,000) vs. 0.9 % (433/50,000) for two samples with Cp values of 30 and 26, respectively. Additionally the assay could generate genome sequences with the percentages of coverage of 94–100 % of 4 different enterovirus serotypes in 73 % of the tested samples, representing the first whole-genome sequences of CV-A6/10/16 from Vietnam, and could assign correctly serotyping results in 100 % of 24 tested specimens. In all but three the obtained consensuses of two replicates from the same sample were 100 % identical, suggesting that our assay is highly reproducible.

Conclusions

In conclusion, we have successfully developed a non-ribosomal rPCR and next-generation sequencing based assay for sensitive detection and direct whole-genome sequencing of HFMD pathogens from clinical samples.

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Title: Development and evaluation of a non-ribosomal random PCR and next-generation sequencing based assay for detection and sequencing of hand, foot and mouth disease pathogens
Authors: Anh To Nguyen
Thanh Tan Tran
Van Minh Tu Hoang
Ngoc My Nghiem
Nhu Nguyen Truc Le
Thanh Thi My Le
Qui Tu Phan
Khanh Huu Truong
Nhan Nguyen Thanh Le
Viet Lu Ho
Viet Chau Do
Tuan Manh Ha
Hung Thanh Nguyen
Chau Van Vinh Nguyen
Guy Thwaites
H. Rogier van Doorn
Tan Van Le
Publication date: 01-12-2016
Publisher: BioMed Central
Published in: Virology Journal / Issue 1/2016
Electronic ISSN: 1743-422X
DOI: https://doi.org/10.1186/s12985-016-0580-9

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Conclusions

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