Development and evaluation of a non-ribosomal random PCR and next-generation sequencing based assay for detection and se
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METHODOLOGY
Open Access
Development and evaluation of a nonribosomal random PCR and nextgeneration sequencing based assay for detection and sequencing of hand, foot and mouth disease pathogens Anh To Nguyen1*, Thanh Tan Tran1, Van Minh Tu Hoang2, Ngoc My Nghiem3, Nhu Nguyen Truc Le1, Thanh Thi My Le3, Qui Tu Phan3, Khanh Huu Truong4, Nhan Nguyen Thanh Le4, Viet Lu Ho2, Viet Chau Do2, Tuan Manh Ha2, Hung Thanh Nguyen4, Chau Van Vinh Nguyen3, Guy Thwaites1,5, H. Rogier van Doorn1,5 and Tan Van Le1
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. Keywords: Hand, foot and mouth disease, Enterovirus A, Random PCR, FR26RV-Endoh primer, Next-generation sequencing
* Correspondence: [email protected] 1 Oxford University Clinical Research Unit, 764 Vo Van Kiet Street, Ward 1, District 5, Ho Chi Minh City, Vietnam Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if c
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