title: An Improved Barcoded Oligonucleotide Primers-based Next-generation Sequencing Approach for Direct Identification of Viral Pathogens in Clinical Specimens.
abstract: OBJECTIVE
  To provide a feasible and cost-effective next-generation sequencing (NGS) method for accurate identification of viral pathogens in clinical specimens, because enormous limitations impede the clinical use of common NGS, such as high cost, complicated procedures, tremendous data analysis, and high background noise in clinical samples.
  
  
  METHODS
  Viruses from cell culture materials or clinical specimens were identified following an improved NGS procedure: reduction of background noise by sample preprocessing, viral enrichment by barcoded oligonucleotide (random hexamer or non-ribosomal hexanucleotide) primer-based amplification, fragmentation-free library construction and sequencing of one-tube mixtures, as well as rapid data analysis using an in-house pipeline.
  
  
  RESULTS
  NGS data demonstrated that both barcoded primer sets were useful to simultaneously capture multiple viral pathogens in cell culture materials or clinical specimens and verified that hexanucleotide primers captured as many viral sequences as hexamers did. Moreover, direct testing of clinical specimens using this improved hexanucleotide primer-based NGS approach provided further detailed genotypes of enteroviruses causing hand, foot, and mouth disease (HFMD) and identified other potential viruses or differentiated misdiagnosis events.
  
  
  CONCLUSIONS
  The improved barcoded oligonucleotide primer-based NGS approach is simplified, time saving, cost effective, and appropriate for direct identification of viral pathogens in clinical practice.
authors.affiliation: Key Laboratory for Medical Virology, National Health and Fam-ily Planning Commission, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
authors.forename: Chun Hua
authors.initials: CH
authors.lastname: Wang
authors.affiliation: Key Laboratory for Medical Virology, National Health and Fam-ily Planning Commission, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
authors.forename: Kai
authors.initials: K
authors.lastname: Nie
authors.affiliation: Key Laboratory for Medical Virology, National Health and Fam-ily Planning Commission, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Key Laboratory for Medical Virology, National Health and Fam-ily Planning Commission, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
authors.forename: Ji
authors.initials: J
authors.lastname: Wang
authors.affiliation: Key Laboratory for Medical Virology, National Health and Fam-ily Planning Commission, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China; Center for Disease Prevention and Control of Hunan Province, Changsha 410005, Hunan, China.
authors.forename: Shuai Feng
authors.initials: SF
authors.lastname: Zhou
authors.affiliation: Key Laboratory for Medical Virology, National Health and Fam-ily Planning Commission, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
authors.forename: Xin Na
authors.initials: XN
authors.lastname: Li
authors.affiliation: Key Laboratory for Medical Virology, National Health and Fam-ily Planning Commission, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
authors.forename: Hang Yu
authors.initials: HY
authors.lastname: Zhou
authors.affiliation: Institute for Viral Disease Control and Prevention, Center for Disease Control and Prevention of Hebei, Shijiazhuang 050000, Hebei, China.
authors.forename: Shun Xiang
authors.initials: SX
authors.lastname: Qi
authors.affiliation: Key Laboratory for Medical Virology, National Health and Fam-ily Planning Commission, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
authors.forename: Xue Jun
authors.initials: XJ
authors.lastname: Ma
mesh_terms: D019411:Clinical Laboratory Techniques; D058893:DNA Barcoding, Taxonomic; D017931:DNA Primers; D004770:Enterovirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D009981:Influenza B virus / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D017423:Sequence Analysis, RNA / Q000379:methods*
chemical_list: D017931:DNA Primers
keywords: Barcoded oligonucleotide primers; Clinical specimen; NGS; Virus identification