title: Rapid single-colony whole-genome sequencing of bacterial pathogens.
abstract: OBJECTIVE
  As a result of the introduction of rapid benchtop sequencers, the time required to subculture a bacterial pathogen to extract sufficient DNA for library preparation can now exceed the time to sequence said DNA. We have eliminated this rate-limiting step by developing a protocol to generate DNA libraries for whole-genome sequencing directly from single bacterial colonies grown on primary culture plates.
  
  
  METHODS
  We developed our protocol using single colonies of 17 bacterial pathogens responsible for severe human infection that were grown using standard diagnostic media and incubation conditions. We then applied this method to four clinical scenarios that currently require time-consuming reference laboratory tests: full identification and genotyping of salmonellae; identification of blaNDM-1, a highly transmissible carbapenemase resistance gene, in Klebsiella pneumoniae; detection of genes encoding staphylococcal toxins associated with specific disease syndromes; and monitoring of vaccine targets to detect vaccine escape in Neisseria meningitidis.
  
  
  RESULTS
  We validated our single-colony whole-genome sequencing protocol for all 40 combinations of pathogen and selective, non-selective or indicator media tested in this study. Moreover, we demonstrated the clinical value of this method compared with current reference laboratory tests.
  
  
  CONCLUSIONS
  This advance will facilitate the implementation of whole-genome sequencing into diagnostic and public health microbiology.
authors.affiliation: Department of Medicine, University of Cambridge, Cambridge, UK.
authors.forename: Claudio U
authors.initials: CU
authors.lastname: Köser
authors.forename: Louise J
authors.initials: LJ
authors.lastname: Fraser
authors.forename: Avgousta
authors.initials: A
authors.lastname: Ioannou
authors.forename: Jennifer
authors.initials: J
authors.lastname: Becq
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Ellington
authors.forename: Matthew T G
authors.initials: MT
authors.lastname: Holden
authors.forename: Sandra
authors.initials: S
authors.lastname: Reuter
authors.forename: M Estée
authors.initials: ME
authors.lastname: Török
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Bentley
authors.forename: Julian
authors.initials: J
authors.lastname: Parkhill
authors.forename: Niall A
authors.initials: NA
authors.lastname: Gormley
authors.forename: Geoffrey P
authors.initials: GP
authors.lastname: Smith
authors.forename: Sharon J
authors.initials: SJ
authors.lastname: Peacock
mesh_terms: D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D016680:Genome, Bacterial; D006801:Humans; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D004269:DNA, Bacterial
keywords: antibiotic resistance; infectious diseases; typing