Capturing diversity of marine heterotrophic protists: one cell at a time

Abstract

Recent applications of culture-independent, molecular methods have revealed unexpectedly high diversity in a variety of functional and phylogenetic groups of microorganisms in the ocean. However, none of the existing research tools are free from significant limitations, such as PCR and cloning biases, low phylogenetic resolution and others. Here, we employed novel, single-cell sequencing techniques to assess the composition of small (o10 lm diameter), heterotrophic protists from the Gulf of Maine. Single cells were isolated by flow cytometry, their genomes amplified, and 18S rRNA marker genes were amplified and sequenced. We compared the results to traditional environmental PCR cloning of sorted cells. The diversity of heterotrophic protists was significantly higher in the library of single amplified genomes (SAGs) than in environmental PCR clone libraries of the 18S rRNA gene, obtained from the same coastal sample. Libraries of SAGs, but not clones contained several recently discovered, uncultured groups, including picobiliphytes and novel marine stramenopiles. Clone, but not SAG, libraries contained several large clusters of identical and nearly identical sequences of Dinophyceae, Cercozoa and Stramenopiles. Similar results were obtained using two alternative primer sets, suggesting that PCR biases may not be the only explanation for the observed patterns. Instead, differences in the number of 18S rRNA gene copies among the various protist taxa probably had a significant role in determining the PCR clone composition. These results show that single-cell sequencing has the potential to more accurately assess protistan community composition than previously established methods. In addition, the creation of SAG libraries opens opportunities for the analysis of multiple genes or entire genomes of the uncultured protist groups.

Patterns and processes in microbial biogeography: do molecules and morphologies give the same answers?

“…Arrows indicate taxa with contribution ⩾ 3% in both approaches (black), only by microscopy (white), or only by HTS (gray). Microscopy results are shown for dimensions compared with the pore size used for sample filtering or primer mismatches is not evident, but there could be other PCR bias (for example, selective amplification of closely-related species), taxon-specific susceptibility to DNA extraction or variability in the copy number of the rDNA operon (Medinger et al, 2010;Heywood et al, 2011). This supports known inconsistencies in relative abundances estimated by HTS and microscopy for ciliates and other protists (Bachy et al, 2013;Egge et al, 2013;Santoferrara et al, 2014;Stoeck et al, 2014).…”

Comparative Analysis of Eukaryotic Marine Microbial Assemblages from 18S rRNA Gene and Gene Transcript Clone Libraries by Using Different Methods of Extraction

“…The picobiliphytes are a recently discovered group of pigmented protists that are related to telonemids, cryptophytes, and katablepharids (11, 34,45). A previous study found that the picobiliphytes may be underrepresented using conventional PCR-based clone libraries (26). Again, a lower gene copy number may have allowed their presence to be masked by the presence of eukaryotes with higher gene copy numbers.…”

Ultrastructural and Single-Cell-Level Characterization Reveals Metabolic Versatility in a Microbial Eukaryote Community from an Ice-Covered Antarctic Lake

“…Traditionally, heterotrophic protists were identified via culture-dependent and microscopic methods (62)(63)(64)(65). Recent studies have shown that a single-cell genomic approach can provide direct evidence of specific predator-prey interactions (66,67). We recovered 22 samples that had high levels of LysoTracker probe-associated fluorescence and were closely related to the stramenopile Pteridomonas danica ( Fig.…”