Development of single-nucleotide polymorphism-based phylum-specific PCR amplification technique: Application to the community analysis using ciliates as a reference organism
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.
Keywordsciliate community analysis phylum-specific PCR pyrosequencing SNP SSU rRNA
Unable to display preview. Download preview PDF.
- Doyle, J.J., and Doyle, J.L. (1987). A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 19, 11–15.Google Scholar
- Hall, T. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 95–98.Google Scholar
- Lynn, D.H. (2008). The ciliated protozoa: characterization, classification, and guide to the literature (New York: Springer).Google Scholar
- Parfrey, L.W., Barbero, E., Lasser, E., Dunthorn, M., Bhattacharya, D., Patterson, D.J., and Katz, L.A. (2006). Evaluating support for the current classification of eukaryotic diversity. PLoS Genet. Genet. 2, 2062–2073.Google Scholar
- Sachidanandam, R., Weissman, D., Schmidt, S.C., Kakol, J.M., Stein, L.D., Marth, G., Sherry, S., Mullikin, J.C., Mortimore, B.J., Willey, D.L., et al. (2001). A map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms. Nature 409, 928–933.PubMedCrossRefGoogle Scholar
- Schloss, P.D., Westcott, S.L., Ryabin, T., Hall, J.R., Hartmann, M., Hollister, E.B., Lesniewski, R.A., Oakley, B.B., Parks, D.H., Robinson, C.J., et al. (2009). Introducing mothur: Open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl. Environ. Microb. 75, 7537–7541.CrossRefGoogle Scholar
- Swofford, D. (2002). PAUP*: phylogenetic analysis using parsimony (* and other methods). Version 4. Sinauer Associates, Sunderland, MA.Google Scholar