Advertisement

Molecules and Cells

, Volume 34, Issue 4, pp 383–391 | Cite as

Development of single-nucleotide polymorphism-based phylum-specific PCR amplification technique: Application to the community analysis using ciliates as a reference organism

  • Jae-Ho Jung
  • Sanghee Kim
  • Seongho Ryu
  • Min-Seok Kim
  • Ye-Seul Baek
  • Se-Joo Kim
  • Joong-Ki Choi
  • Joong-Ki Park
  • Gi-Sik Min
Article

Abstract

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.

Keywords

ciliate community analysis phylum-specific PCR pyrosequencing SNP SSU rRNA 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman, D.J. (1990). Basic local alignment search tool. J. Mol. Biol. 215, 403–410.PubMedGoogle Scholar
  2. Blackwood, C.B., Oaks, A., and Buyers, J.S. (2005). Phylum- and class-specific PCR primers for general microbial community analysis. Appl. Environ. Microb. 71, 6193–6198.CrossRefGoogle Scholar
  3. Bui, M., and Liu, Z. (2009). Simple allele-discriminating PCR for cost-effective and rapid genotyping and mapping. Plant Methods 5, 1.PubMedCrossRefGoogle Scholar
  4. Carrino-Kyker, S.R., and Swanson, A.K. (2008). Temporal and spatial patterns of eukaryotic and bacterial communities found in vernal pools. Appl. Environ. Microb. 74, 2554–2557.CrossRefGoogle Scholar
  5. Dopheide, A., Lear, G., Stott, R., and Lewis, G. (2008). Molecular characterization of ciliate diversity in stream biofilms. Appl. Environ. Microb. 74, 1740–1747.CrossRefGoogle Scholar
  6. 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
  7. Guindon, S., and Gascuel, O. (2003). A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol. 52, 696–704.PubMedCrossRefGoogle Scholar
  8. Haliassos, A., Chomel, J.C., Grandjouan, S., Kruh, J., Kaplan, J.C., and Kitzis, A. (1989). Detection of minority point mutations by modified PCR technique: a new approach for a sensitive diagnosis of tumor-progression markers. Nucleic Acids Res. 17, 8093–8099.PubMedCrossRefGoogle Scholar
  9. 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
  10. Holzmann, M., Habura, A., Giles, H., Bowser, S.S., and Pawlowski, J. (2003). Freshwater foraminiferans revealed by analysis of environmental DNA samples. J. Eukaryot. Microbiol. 50, 135–139.PubMedCrossRefGoogle Scholar
  11. Huber, T., Faulkner, G., and Hugenholtz, P. (2004). Bellerophon: a program to detect chimeric sequences in multiple sequence alignments. Bioinformatics 20, 2317–2319.PubMedCrossRefGoogle Scholar
  12. Huson, D.H., Richter, D.C., Mitra, S., Auch, A.F., and Schuster, S.C. (2009). Methods for comparative metagenomics. BMC Bioinformatics 10, S12.PubMedCrossRefGoogle Scholar
  13. Konieczny, A., and Ausubel, F.M. (1993). A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based markers. Plant J. 4, 403–410.PubMedCrossRefGoogle Scholar
  14. Kumar, S., Tamura, K., and Nei, M. (2004). MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief. Bioinform. 5, 150–163.PubMedCrossRefGoogle Scholar
  15. Lahr, D.J.G., and Katz, L.A. (2009). Reducing the impact of PCRmediated recombination in molecular evolution and environmental studies using a new-generation high-fidelity DNA polymerase. Biotechniques 47, 857–863.PubMedGoogle Scholar
  16. Lin, S.J., Zhang, H., Hou, Y., Miranda, L., and Bhattacharya, D. (2006). Development of a dinoflagellate-oriented PCR primer set leads to detection of picoplanktonic dinoflagellates from long island sound. Appl. Environ. Microb. 72, 5626–5630.CrossRefGoogle Scholar
  17. Lynn, D.H. (2003). Morphology or molecules: how do we identify the major lineages of ciliates (Phylum Ciliophora)? Eur. J. Protistol. 39, 356–364.CrossRefGoogle Scholar
  18. Lynn, D.H. (2008). The ciliated protozoa: characterization, classification, and guide to the literature (New York: Springer).Google Scholar
  19. Mashayekhi, F., and Ronaghi, M. (2007). Analysis of read length limiting factors in Pyrosequencing chemistry. Anal. Biochem. 363, 275–287.PubMedCrossRefGoogle Scholar
  20. Medlin, L., Elwood, H.J., Stickel, S., and Sogin, M.L. (1988). The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions. Gene 71, 491–499.PubMedCrossRefGoogle Scholar
  21. Moon-van der Staay, S.Y., De Wachter, R., and Vaulot, D. (2001). Oceanic 18S rDNA sequences from picoplankton reveal unsuspected eukaryotic diversity. Nature 409, 607–610.PubMedCrossRefGoogle Scholar
  22. Morita, A., Nakayama, T., Doba, N., Hinohara, S., Mizutani, T., and Soma, M. (2007). Genotyping of triallelic SNPs using TaqMan® PCR. Mol. Cell. Probes 21, 171–176.PubMedCrossRefGoogle Scholar
  23. Nolte, V., Pandey, R.V., Jost, S., Medinger, R., Ottenwalder, B., Boenigk, J., and Schlotterer, C. (2010). Contrasting seasonal niche separation between rare and abundant taxa conceals the extent of protist diversity. Mol. Ecol. 19, 2908–2915.PubMedCrossRefGoogle Scholar
  24. Nossa, C.W., Oberdorf, W.E., Yang, L.Y., Aas, J.A., Paster, B.J., DeSantis, T.Z., Brodie, E.L., Malamud, D., Poles, M.A., and Pei, Z.H. (2010). Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome. World J. Gastroenterol. 16, 4135–4144.PubMedCrossRefGoogle Scholar
  25. 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
  26. Posada, D., and Crandall, K.A. (1998). MODELTEST: testing the model of DNA substitution. Bioinformatics 14, 817–818.PubMedCrossRefGoogle Scholar
  27. Pruesse, E., Quast, C., Knittel, K., Fuchs, B.M., Ludwig, W.G., Peplies, J., and Glockner, F.O. (2007). SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res. 35, 7188–7196.PubMedCrossRefGoogle Scholar
  28. Quince, C., Lanzen, A., Davenport, R.J., and Turnbaugh, P.J. (2011). Removing noise from pyrosequenced amplicons. BMC Bioinformatics 12, 38.PubMedCrossRefGoogle Scholar
  29. Richards, T.A., and Bass, D. (2005). Molecular screening of freeliving microbial eukaryotes: diversity and distribution using a meta-analysis. Curr. Opin. Microbiol. 8, 240–252.PubMedCrossRefGoogle Scholar
  30. Richards, T.A., Vepritskiy, A.A., Gouliamova, D.E., and Nierzwicki-Bauer, S.A. (2005). The molecular diversity of freshwater picoeukaryotes from an oligotrophic lake reveals diverse, distinctive and globally dispersed lineages. Environ. Microbiol. 7, 1413–1425.PubMedCrossRefGoogle Scholar
  31. 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
  32. Scheckenbach, F., Hausmann, K., Wylezich, C., Weitere, M., and Arndt, H. (2010). Large-scale patterns in biodiversity of microbial eukaryotes from the abyssal sea floor. Proc. Natl. Acad. Sci. USA 107, 115–120.PubMedCrossRefGoogle Scholar
  33. 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
  34. Shanks, O.C., Kelty, C.A., Archibeque, S., Jenkins, M., Newton, R.J., McLellan, S.L., Huse, S.M., and Sogin, M.L. (2011). Community structures of fecal bacteria in cattle from different animal feeding operations. Appl. Environ. Microbiol. 77, 2992–3001.PubMedCrossRefGoogle Scholar
  35. Stoeck, T., Behnke, A., Christen, R., Amaral-Zettler, L., Rodriguez-Mora, M.J., Chistoserdov, A., Orsi, W., and Edgcomb, V.P. (2009). Massively parallel tag sequencing reveals the complexity of anaerobic marine protistan communities. BMC Biol. 7, 72.PubMedCrossRefGoogle Scholar
  36. Stoeck, T., Bass, D., Nebel, M., Christen, R., Jones, M.D.M., Breiner, H.-W., and Richards, T.A. (2010). Multiple marker parallel tag environmental DNA sequencing reveals a highly complex eukaryotic community in marine anoxic water. Mol. Ecol. 19, 21–31.PubMedCrossRefGoogle Scholar
  37. Swofford, D. (2002). PAUP*: phylogenetic analysis using parsimony (* and other methods). Version 4. Sinauer Associates, Sunderland, MA.Google Scholar
  38. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., and Higgins, D.G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876–4882.PubMedCrossRefGoogle Scholar
  39. Zou, F., Lee, S., Knowles, M.R., and Wright, F.A. (2010). Quantification of population structure using correlated SNPs by shrinkage principal components. Hum. Hered. 70, 9–22.PubMedCrossRefGoogle Scholar

Copyright information

© The Korean Society for Molecular and Cellular Biology and Springer Netherlands 2012

Authors and Affiliations

  • Jae-Ho Jung
    • 1
  • Sanghee Kim
    • 2
  • Seongho Ryu
    • 3
  • Min-Seok Kim
    • 1
  • Ye-Seul Baek
    • 1
  • Se-Joo Kim
    • 1
    • 6
  • Joong-Ki Choi
    • 4
  • Joong-Ki Park
    • 5
  • Gi-Sik Min
    • 1
  1. 1.Department of Biological SciencesInha UniversityIncheonKorea
  2. 2.Korea Polar Research Institute (KORDI)IncheonKorea
  3. 3.Department of Cell and Developmental BiologyWeill Cornell Medical CollegeNew YorkUSA
  4. 4.Department of OceanographyInha UniversityIncheonKorea
  5. 5.Department of Parasitology and Graduate Program in Cell Biology and Genetics, College of MedicineChungbuk National UniversityCheongjuKorea
  6. 6.Deep-sea and Seabed Resources Research DivisionKorea Institute of Ocean Science and TechnologyAnsanKorea

Personalised recommendations