title: Deep sequencing analyses of low density microbial communities: working at the boundary of accurate microbiota detection.
abstract: BACKGROUND
  Accurate analyses of microbiota composition of low-density communities (10(3)-10(4) bacteria/sample) can be challenging. Background DNA from chemicals and consumables, extraction biases as well as differences in PCR efficiency can significantly interfere with microbiota assessment. This study was aiming to establish protocols for accurate microbiota analysis at low microbial density.
  
  
  METHODS
  To examine possible effects of bacterial density on microbiota analyses we compared microbiota profiles of serial diluted saliva and low (nares, nasopharynx) and high-density (oropharynx) upper airway communities in four healthy individuals. DNA was extracted with four different extraction methods (Epicentre Masterpure, Qiagen DNeasy, Mobio Powersoil and a phenol bead-beating protocol combined with Agowa-Mag-mini). Bacterial DNA recovery was analysed by 16S qPCR and microbiota profiles through GS-FLX-Titanium-Sequencing of 16S rRNA gene amplicons spanning the V5-V7 regions.
  
  
  RESULTS
  Lower template concentrations significantly impacted microbiota profiling results. With higher dilutions, low abundant species were overrepresented. In samples of <10(5) bacteria per ml, e.g. DNA <1 pg/µl, microbiota profiling deviated from the original sample and other dilutions showing a significant increase in the taxa Proteobacteria and decrease in Bacteroidetes. In similar low density samples, DNA extraction method determined if DNA levels were below or above 1 pg/µl and, together with lysis preferences per method, had profound impact on microbiota analyses in both relative abundance as well as representation of species.
  
  
  CONCLUSIONS
  This study aimed to interpret microbiota analyses of low-density communities. Bacterial density seemed to interfere with microbiota analyses at < than 10(6) bacteria per ml or DNA <1 pg/µl. We therefore recommend this threshold for working with low density materials. This study underlines that bias reduction is crucial for adequate profiling of especially low-density bacterial communities.
authors.affiliation: Department of Pediatric Infectious Diseases and Immunity UMC Utrecht, Utrecht, The Netherlands.
authors.forename: Giske
authors.initials: G
authors.lastname: Biesbroek
authors.forename: Elisabeth A M
authors.initials: EA
authors.lastname: Sanders
authors.forename: Guus
authors.initials: G
authors.lastname: Roeselers
authors.forename: Xinhui
authors.initials: X
authors.lastname: Wang
authors.forename: Martien P M
authors.initials: MP
authors.lastname: Caspers
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Trzciński
authors.forename: Debby
authors.initials: D
authors.lastname: Bogaert
authors.forename: Bart J F
authors.initials: BJ
authors.lastname: Keijser
mesh_terms: D058491:Bacterial Load; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D054884:Host-Pathogen Interactions; D006801:Humans; D054892:Metagenome / Q000235:genetics*; D009305:Nasopharynx / Q000382:microbiology; D012137:Respiratory System / Q000382:microbiology
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal