title: Environmental rRNA inventories miss over half of protistan diversity.
abstract: BACKGROUND
  The main tool to discover novel microbial eukaryotes is the rRNA approach. This approach has important biases, including PCR discrimination against certain rRNA gene species, which makes molecular inventories skewed relative to the source communities. The degree of this bias has not been quantified, and it remains unclear whether species missed from clone libraries could be recovered by increasing sequencing efforts, or whether they cannot be detected in principle. Here we attempt to discriminate between these possibilities by statistically analysing four protistan inventories obtained using different general eukaryotic PCR primers.
  
  
  RESULTS
  We show that each PCR primer set-specific clone library is not a sample from the community diversity but rather from a fraction of this diversity. Therefore, even sequencing such clone libraries to saturation would only recover that fraction, which, according to the parametric models, varies between 17 +/- 4% to 49 +/- 10%, depending on the set of primers. The pooled data is thus qualitatively richer than individual libraries, even if normalized to the same sequencing effort.
  
  
  CONCLUSIONS
  The use of a single pair of primers leads to significant underestimation of the true community richness at all levels of taxonomic hierarchy. The majority of available protistan rRNA gene surveys likely sampled less than half of the target diversity, and might have completely missed the rest. The use of multiple PCR primers reduces this bias but does not necessarily eliminate it.
authors.affiliation: Department of Environmental Science, Kangwon National University, Kangwon-Do, Korea. sunokjeon@kangwon.ac.kr
authors.forename: Sunok
authors.initials: S
authors.lastname: Jeon
authors.forename: John
authors.initials: J
authors.lastname: Bunge
authors.forename: Chesley
authors.initials: C
authors.lastname: Leslin
authors.forename: Thorsten
authors.initials: T
authors.lastname: Stoeck
authors.forename: Sunhee
authors.initials: S
authors.lastname: Hong
authors.forename: Slava S
authors.initials: SS
authors.lastname: Epstein
mesh_terms: D000818:Animals; D017931:DNA Primers / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics*; D004275:DNA, Ribosomal / Q000235:genetics; D056890:Eukaryota / Q000235:genetics*; D014644:Genetic Variation; D010802:Phylogeny; D012335:RNA, Ribosomal / Q000235:genetics*; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D012335:RNA, Ribosomal