Sun-Hee Hong

Polymerase chain reaction primers miss half of rRNA microbial diversity.

The rRNA approach is the principal tool to study microbial diversity, but it has important biases. These include polymerase chain reaction (PCR) primers bias, and relative inefficiency of DNA extraction techniques. Such sources of potential undersampling of microbial diversity are well known, but the scale of the undersampling has not been quantified. Using a marine tidal flat bacterial community as a model, we show that even with unlimited sampling and sequencing effort, a single combination of PCR primers/DNA extraction technique enables theoretical recovery of only half of the richness recoverable with three such combinations. This shows that different combinations of PCR primers/DNA extraction techniques recover in principle different species, as well as higher taxa. The majority of earlier estimates of microbial richness seem to be underestimates. The combined use of multiple PCR primer sets, multiple DNA extraction techniques, and deep community sequencing will minimize the biases and recover substantially more species than prior studies, but we caution that even this—yet to be used—approach may still leave an unknown number of species and higher taxa undetected.

Environmental rRNA inventories miss over half of protistan diversity

BackgroundThe 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.ResultsWe 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.ConclusionThe 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.

Synthetic Statistical Approach Reveals a High Degree of Richness of Microbial Eukaryotes in an Anoxic Water Column

ABSTRACT Molecular surveys suggest that communities of microbial eukaryotes are remarkably rich, because even large clone libraries seem to capture only a minority of species. This provides a qualitative picture of protistan richness but does not measure its real extent either locally or globally. Statistical analysis can estimate a communitys richness, but the specific methods used to date are not always well grounded in statistical theory. Here we study a large protistan molecular survey from an anoxic water column in the Cariaco Basin (Caribbean Sea). We group individual 18S rRNA gene sequences into operational taxonomic units (OTUs) using different cutoff values for sequence similarity (99 to 50%) and systematically apply parametric models and nonparametric estimators to the OTU frequency data to estimate the total protistan diversity. The parametric models provided statistically sound estimates of protistan richness, with biologically meaningful standard errors, maximal data usage, and extensive model diagnostics and were preferable to the available nonparametric tools. Our clone library exceeded 700 clones but still covered only a minority of species and less than half of the larger protistan clades. Our estimates of total protistan richness portray the target community as very rich at all OTU levels, with hundreds of different populations apparently co-occurring in the small (3-liter) volume of our sample, as well as dozens of clades of the highest taxonomic order. These estimates are among the first for microbial eukaryotes that are obtained using state-of-the-art statistical methods and can serve as benchmark numbers for the local diversity of protists.

A novel archaeal group in the phylum Crenarchaeota found unexpectedly in an eukaryotic survey in the Cariaco Basin

Archaea have been found in many more diverse habitats than previously believed due in part to modern molecular approaches to discovering microbial diversity. We report here an unexpected expansion of the habitat diversity of the Archaea in the Cariaco Basin we found using a primer set designed for 18S eukaryotic rDNA sequence analysis. The results presented here expand the originally identified 9 archaeal clones reported in this environment using bacterial/archaeal primers to 152 archaeal clones: 67 (18 OTU) of these clones were found at a depth of 900 m of station A while 71 (9 OTU) of them were at a depth of between 300∼335 m of station B&C depending upon which location the samples were taken. We used three phylogenetic analysis methods and detected 20 phylotypes belonging to a single previously unreported group distantly related to the Crenarchaeota. Also, we determined that the original nine sequences did not fall into any of the known phyla of the Archaea suggesting that they may represent a novel group within the Kingdom Archaea. Thus, from these two studies, we suggest that Archaea in the Cariaco Basin could be unique; however, further studies using archaeal-specific primers and the design of new primers as well as the systematic use of several different primer combinations may improve the chances of understanding the archeal diversity in the Cariaco Basin.

Analysis of the bacterial community in Lake Soyang using an in situ hybridization method

Recently, molecular techniques have being applied to bacterial identification in situ, such as DNA sequencing with PCR, DNA-DNA hybridizat~on and single cell idenrification using rRNA-spectfic probes. 5uch methods have provided direct informatian regarding bacterial communiry and environmental conditions. Among these molecular techniques, in situ hybridization with a group-specific fluorescent-labeled probe is simple and quick to apply (AMANN er al. 1995). Group-specific and 165 and 235 rRNA-targeted probe molecules are ideal targets for whole cell hybridization because they are conserved molecules and comprise both higher and lower evolurionary conservation (WAGNER et al. 1993). More than 80% of bacteria are Gram negative in a freshwater environment (KANG & 5EKI 1983), and phylogenetically, many Gram-negative bacteria fali into the class Proteobacteria, which is subdivided into a-, ~-, yand Õ-subclasses (DE LAY 1991). Flavobacterium is also abundant in freshwater (RHEINHE!MER 1980). The aim of this study was to define the temporal and spatial changes of bacterial communities by using the in si tu hybridization probes, which are specific for al! members of the domain Bacteria, for rhe a-, ~-, and y-subclasses of the class Proteobacteria, and for the Cytophaga-Flavobacterium group.