Despite recent advance in mass sequencing technologies such as pyrosequencing, assessment of culture-independent microbial eukaryote community structures using universal primers remains very difficult due to the tremendous richness and complexity of organisms in these communities. Use of a specific PCR marker targeting a particular group would provide enhanced sensitivity and more in-depth evaluation of microbial eukaryote communities compared to what can be achieved with universal primers. We discovered that many phylum- or groupspecific single-nucleotide polymorphisms (SNPs) exist in small subunit ribosomal RNA (SSU rRNA) genes from diverse eukaryote groups. By applying this discovery to a known simple allele-discriminating (SAP) PCR method, we developed a technique that enables the identification of organisms belonging to a specific higher taxonomic group (or phylum) among diverse types of eukaryotes. We performed an assay using two complementary methods, pyrosequencing and clone library screening. In doing this, specificities for the group (ciliates) targeted in this study in bulked environmental samples were 94.6% for the clone library and 99.2% for pyrosequencing, respectively. In particular, our novel technique showed high selectivity for rare species, a feature that may be more important than the ability to identify quantitatively predominant species in community structure analyses. Additionally, our data revealed that a target-specific library (or ciliate-specific one for the present study) can better explain the ecological features of a sampling locality than a universal library.
Bibliographical noteFunding Information:
We thank Dr. Rob DeSalle and Dr. Mark Siddall (American Museum of Natural History) for their v aluable input and comments on earlier drafts of this manuscript. This work was partially supported by the Basic Science Research Program (2012R1A1A2006835) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (G.-S. Min), and the Basic Research Program of the Korea Polar Research Institute project (PE12030; S. Kim).
- Community analysis
- Phylum-specific PCR
- SSU rRNA