title: The use of next generation sequencing in the diagnosis and typing of respiratory infections.
abstract: BACKGROUND
  Molecular assays are the gold standard methods used to diagnose viral respiratory pathogens. Pitfalls associated with this technique include limits to the number of targeted pathogens, the requirement for continuous monitoring to ensure sensitivity/specificity is maintained and the need to evolve to include emerging pathogens. Introducing target independent next generation sequencing (NGS) could resolve these issues and revolutionise respiratory viral diagnostics.
  
  
  OBJECTIVE
  To compare the sensitivity and specificity of target independent NGS against the current standard diagnostic test.
  
  
  METHODS
  Diagnostic RT-PCR of clinical samples was carried out in parallel with target independent NGS. NGS sequences were analyzed to determine the proportion with viral origin and consensus sequences were used to establish viral genotypes and serotypes where applicable.
  
  
  RESULTS
  89 nasopharyngeal swabs were tested. A viral pathogen was detected in 43% of samples by NGS and 54% by RT-PCR. All NGS viral detections were confirmed by RT-PCR.
  
  
  CONCLUSIONS
  Target independent NGS can detect viral pathogens in clinical samples. Where viruses were detected by RT-PCR alone the Ct value was higher than those detected by both assays, suggesting an NGS detection cut-off - Ct=32. The sensitivity and specificity of NGS compared with RT-PCR was 78% and 80% respectively. This is lower than current diagnostic assays but NGS provided full genome sequences in some cases, allowing determination of viral subtype and serotype. Sequencing technology is improving rapidly and it is likely that within a short period of time sequencing depth will increase in-turn improving test sensitivity.
authors.affiliation: MRC-University of Glasgow Centre for Virus Research, Garscube Campus, 464 Bearsden Road, Glasgow, G61 1QH, UK. Electronic address: Fiona_thorburn@hotmail.com.
authors.forename: Fiona
authors.initials: F
authors.lastname: Thorburn
authors.affiliation: West Of Scotland Specialist Virology Centre, Glasgow Royal Infirmary, Alexandra Parade, Glasgow, G31 2ER, UK.
authors.forename: Susan
authors.initials: S
authors.lastname: Bennett
authors.affiliation: MRC-University of Glasgow Centre for Virus Research, Garscube Campus, 464 Bearsden Road, Glasgow, G61 1QH, UK.
authors.forename: Sejal
authors.initials: S
authors.lastname: Modha
authors.affiliation: Department of Pathology, University of Otago, Christchurch, New Zealand.
authors.forename: David
authors.initials: D
authors.lastname: Murdoch
authors.affiliation: West Of Scotland Specialist Virology Centre, Glasgow Royal Infirmary, Alexandra Parade, Glasgow, G31 2ER, UK.
authors.forename: Rory
authors.initials: R
authors.lastname: Gunson
authors.affiliation: MRC-University of Glasgow Centre for Virus Research, Garscube Campus, 464 Bearsden Road, Glasgow, G61 1QH, UK.
authors.forename: Pablo R
authors.initials: PR
authors.lastname: Murcia
mesh_terms: D004279:DNA, Viral / Q000032:analysis; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009305:Nasopharynx / Q000821:virology; D010865:Pilot Projects; D012367:RNA, Viral / Q000032:analysis; D012141:Respiratory Tract Infections / Q000175:diagnosis / Q000821:virology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D014777:Virus Diseases / Q000175:diagnosis*; D014780:Viruses / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D004279:DNA, Viral; D012367:RNA, Viral
keywords: Diagnostics; Next generation sequencing; Viral respiratory infection; Virus detection;