title: Sterile paper points as a bacterial DNA-contamination source in microbiome profiles of clinical samples.
abstract: OBJECTIVE
  High throughput sequencing of bacterial DNA from clinical samples provides untargeted, open-ended information on the entire microbial community. The downside of this approach is the vulnerability to DNA contamination from other sources than the clinical sample. Here we describe contamination from sterile paper points (PPs) used in microbial sample collection.
  
  
  METHODS
  Peri-implant samples from 48 individuals were collected using sterile PPs. Control samples contained only PPs or DNA extraction blank controls. 16S rRNA gene libraries were sequenced using 454 pyrosequencing. 16S rRNA gene copy numbers were measured by quantitative PCR.
  
  
  RESULTS
  Nearly half of the sequencing reads belonged to two OTUs classified as Enterococcus (25% of reads) or Exiguobacterium (21%), which are not typical oral microorganisms. Of 87 peri-implant samples, only 10 samples (11%) contained neither of the two OTUs. The relative abundance of both unusual OTUs correlated with each other (p<0.001; r=0.828, Spearman correlation). The control samples showed that 2 of 4 (50%) of the sterile unused PPs contained bacterial DNA equivalent to 1.2 × 10(3) and 1.1 × 10(4) cells respectively, which was within the range of DNA in the clinical samples (average 1.8 × 10(7), SD 4.8 × 10(7), min 4.4 × 10(2), max 2.8 × 10(8)). The microbial profile from these PPs was dominated (>83% of reads) by the two unusual OTUs.
  
  
  CONCLUSIONS
  Sterile PPs can contain contaminating bacterial DNA. The use of PPs as a sampling tool for microbial profiling of clinical samples by open-ended techniques such as sequencing or DGGE should be avoided.
  
  
  CONCLUSIONS
  Clinicians working with PPs as sampling tools for bacterial DNA should consider using an alternative sampling tool, because sterile unused PPs can be a considerable source of foreign bacterial DNA. We recommend sterile curettes for collecting clinical samples for open-ended techniques, such as sequencing or DGGE.
authors.affiliation: Department of Oral Function and Restorative Dentistry, Section of Oral Implantology and Prosthodontics, Research Institute MOVE, Academic Centre for Dentistry Amsterdam (ACTA), Gustav Mahlerlaan 3004, 1081 LA Amsterdam, The Netherlands. Electronic address: jhorst@acta.nl.
authors.forename: Joyce
authors.initials: J
authors.lastname: van der Horst
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Buijs
authors.forename: Marja L
authors.initials: ML
authors.lastname: Laine
authors.forename: Daniël
authors.initials: D
authors.lastname: Wismeijer
authors.forename: Bruno G
authors.initials: BG
authors.lastname: Loos
authors.forename: Wim
authors.initials: W
authors.lastname: Crielaard
authors.forename: Egija
authors.initials: E
authors.lastname: Zaura
mesh_terms: D056449:Bacillales / Q000235:genetics; D003430:Cross-Sectional Studies; D059018:DNA Contamination; D004269:DNA, Bacterial / Q000032:analysis*; D015921:Dental Implants / Q000382:microbiology; D003773:Dental Plaque / Q000382:microbiology; D016983:Enterococcus / Q000235:genetics; D004866:Equipment Contamination; D018628:Gene Dosage; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008422:Materials Testing; D064307:Microbiota; D010209:Paper; D010514:Periodontal Pocket / Q000382:microbiology; D012336:RNA, Ribosomal, 16S / Q000032:analysis; D017422:Sequence Analysis, DNA
chemical_list: D004269:DNA, Bacterial; D015921:Dental Implants; D012336:RNA, Ribosomal, 16S
keywords: 16S rRNA gene sequencing; Contamination; DNA; Microbiome; Paper point