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.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Oxford University Clinical Research Unit, 764 Vo Van Kiet Street, Ward 1, District 5, Ho Chi Minh City, Vietnam. anhnt@oucru.org.
authors.forename: Anh To
authors.initials: AT
authors.lastname: Nguyen
authors.affiliation: Oxford University Clinical Research Unit, 764 Vo Van Kiet Street, Ward 1, District 5, Ho Chi Minh City, Vietnam.
authors.forename: Thanh Tan
authors.initials: TT
authors.lastname: Tran
authors.affiliation: Children's Hospital 2, Ho Chi Minh City, Vietnam.
authors.forename: Van Minh Tu
authors.initials: VM
authors.lastname: Hoang
authors.affiliation: Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam.
authors.forename: Ngoc My
authors.initials: NM
authors.lastname: Nghiem
authors.affiliation: Oxford University Clinical Research Unit, 764 Vo Van Kiet Street, Ward 1, District 5, Ho Chi Minh City, Vietnam.
authors.forename: Nhu Nguyen Truc
authors.initials: NN
authors.lastname: Le
authors.affiliation: Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam.
authors.forename: Thanh Thi My
authors.initials: TT
authors.lastname: Le
authors.affiliation: Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam.
authors.forename: Qui Tu
authors.initials: QT
authors.lastname: Phan
authors.affiliation: Children's Hospital 1, Ho Chi Minh City, Vietnam.
authors.forename: Khanh Huu
authors.initials: KH
authors.lastname: Truong
authors.affiliation: Children's Hospital 1, Ho Chi Minh City, Vietnam.
authors.forename: Nhan Nguyen Thanh
authors.initials: NN
authors.lastname: Le
authors.affiliation: Children's Hospital 2, Ho Chi Minh City, Vietnam.
authors.forename: Viet Lu
authors.initials: VL
authors.lastname: Ho
authors.affiliation: Children's Hospital 2, Ho Chi Minh City, Vietnam.
authors.forename: Viet Chau
authors.initials: VC
authors.lastname: Do
authors.affiliation: Children's Hospital 2, Ho Chi Minh City, Vietnam.
authors.forename: Tuan Manh
authors.initials: TM
authors.lastname: Ha
authors.affiliation: Children's Hospital 1, Ho Chi Minh City, Vietnam.
authors.forename: Hung Thanh
authors.initials: HT
authors.lastname: Nguyen
authors.affiliation: Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam.
authors.forename: Chau Van Vinh
authors.initials: CV
authors.lastname: Nguyen
authors.affiliation: Oxford University Clinical Research Unit, 764 Vo Van Kiet Street, Ward 1, District 5, Ho Chi Minh City, Vietnam.|Centre for Tropical Medicine, Nuffield Department of Medicine, University of Oxford, Oxford, UK.
authors.forename: Guy
authors.initials: G
authors.lastname: Thwaites
authors.affiliation: Oxford University Clinical Research Unit, 764 Vo Van Kiet Street, Ward 1, District 5, Ho Chi Minh City, Vietnam.|Centre for Tropical Medicine, Nuffield Department of Medicine, University of Oxford, Oxford, UK.
authors.forename: H Rogier
authors.initials: HR
authors.lastname: van Doorn
authors.affiliation: Oxford University Clinical Research Unit, 764 Vo Van Kiet Street, Ward 1, District 5, Ho Chi Minh City, Vietnam.
authors.forename: Tan Van
authors.initials: TV
authors.lastname: Le
mesh_terms: D029821:Enterovirus A, Human / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D005838:Genotype; D006232:Hand, Foot and Mouth Disease / Q000175:diagnosis / Q000821:virology*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D012703:Serotyping
keywords: Enterovirus A; FR26RV-Endoh primer; Hand, foot and mouth disease; Next-generation sequencing; Random PCR