title: Implications of multiple freeze-thawing on respiratory samples for culture-independent analyses.
abstract: BACKGROUND
  Best practice when performing culture-independent microbiological analysis of sputum samples involves their rapid freezing and storage at -80°C. However, accessing biobanked collections can mean that material has been passed through repeated freeze-thaw cycles. The aim of this study was to determine the impact of these cycles on microbial community profiles.
  
  
  METHODS
  Sputum was collected from eight adults with cystic fibrosis, and each sample was subjected to six freeze-thaw cycles. Following each cycle, an aliquot was removed and treated with propidium monoazide (PMA) prior to DNA extraction and 16S rRNA gene pyrosequencing.
  
  
  RESULTS
  The impact of freeze-thaw cycles was greatest on rare members of the microbiota, with variation beyond that detected with within-sample repeat analysis observed after three cycles.
  
  
  CONCLUSIONS
  Four or more freeze thaw cycles result in a significant distortion of microbiota profiles from CF sputum.
authors.affiliation: NERC Centre for Ecology & Hydrology, Wallingford, OX10 8BB, UK; Institute of Pharmaceutical Science, Molecular Microbiology Research Laboratory, King's College London, London SE1 9NH, UK.
authors.forename: Leah
authors.initials: L
authors.lastname: Cuthbertson
authors.affiliation: SAHMRI Infection and Immunity Theme, Flinders University, Adelaide, Australia.
authors.forename: Geraint B
authors.initials: GB
authors.lastname: Rogers
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK; Rowett Institute of Nutrition and Health, Aberdeen AB21 9SB, UK.
authors.forename: Alan W
authors.initials: AW
authors.lastname: Walker
authors.affiliation: NERC Centre for Ecology & Hydrology, Wallingford, OX10 8BB, UK.
authors.forename: Anna
authors.initials: A
authors.lastname: Oliver
authors.affiliation: Department of Pediatrics, University of Washington, Box 356320, HSB K328A, Seattle, WA 98105, USA; Department of Microbiology, University of Washington, Box 356320, HSB K328A, Seattle, WA 98105, USA.
authors.forename: Lucas R
authors.initials: LR
authors.lastname: Hoffman
authors.affiliation: Cystic Fibrosis Unit, Southampton University Hospitals NHS Trust, Southampton SO16 6YD, UK.
authors.forename: Mary P
authors.initials: MP
authors.lastname: Carroll
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK.
authors.forename: Julian
authors.initials: J
authors.lastname: Parkhill
authors.affiliation: Institute of Pharmaceutical Science, Molecular Microbiology Research Laboratory, King's College London, London SE1 9NH, UK.
authors.forename: Kenneth D
authors.initials: KD
authors.lastname: Bruce
authors.affiliation: NERC Centre for Ecology & Hydrology, Wallingford, OX10 8BB, UK. Electronic address: cjvdg@ceh.ac.uk.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: van der Gast
mesh_terms: D000328:Adult; D018070:Biological Specimen Banks; D015925:Cryopreservation / Q000379:methods*; D003550:Cystic Fibrosis / Q000382:microbiology*; D006801:Humans; D064307:Microbiota; D013048:Specimen Handling / Q000379:methods*; D013183:Sputum / Q000382:microbiology*
keywords: Biobank; Microbiome; Microbiota; Propidium monoazide; Pyrosequencing; Sputum