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Integrating High-Throughput Pyrosequencing and Quantitative Real-Time PCR to Analyze Complex Microbial Communities

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High-Throughput Next Generation Sequencing

Part of the book series: Methods in Molecular Biology ((MIMB,volume 733))

Abstract

New high-throughput technologies continue to emerge for studying complex microbial communities. In particular, massively parallel pyrosequencing enables very high numbers of sequences, providing a more complete view of community structures and a more accurate inference of the functions than has been possible just a few years ago. In parallel, quantitative real-time PCR (QPCR) allows quantitative monitoring of specific community members over time, space, or different environmental conditions. In this review, we discuss the principles of these two methods and their complementary applications in studying microbial ecology in bioenvironmental systems. We explain parallel sequencing of amplicon libraries and using bar codes to differentiate multiple samples in a pyrosequencing run. We also describe best procedures and chemistries for QPCR amplifications and address advantages of applying automation to increase accuracy. We provide three examples in which we used pyrosequencing and QPCR together to define and quantify members of microbial communities: in the human large intestine, in a methanogenic digester whose sludge was made more bioavailable by a high-voltage pretreatment, and on the biofilm anode of a microbial electrolytic cell. We highlight our key findings in these systems and how both methods were used in concert to achieve those findings. Finally, we supply detailed methods for generating PCR amplicon libraries for pyrosequencing, pyrosequencing data analysis, QPCR methodology, instrumentation, and automation.

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Author information

Authors and Affiliations

  1. Center for Environmental Biotechnology, Biodesign Institute, Arizona State University, Tempe, AZ, USA

    Rosa Krajmalnik-Brown

Authors
  1. Husen Zhang
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  2. Prathap Parameswaran
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  3. Jonathan Badalamenti
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  4. Bruce E. Rittmann
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  5. Rosa Krajmalnik-Brown
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Corresponding author

Correspondence to Rosa Krajmalnik-Brown .

Editor information

Editors and Affiliations

  1. , Department of Poultry Science, University of Arkansas, W. Maple St. 1260, Fayetteville, 72701, Arkansas, USA

    Young Min Kwon

  2. Department of Food Science, University of Arkansas, N. Young Ave. 2650, Fayetteville, 72704, Arkansas, USA

    Steven C. Ricke

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Zhang, H., Parameswaran, P., Badalamenti, J., Rittmann, B.E., Krajmalnik-Brown, R. (2011). Integrating High-Throughput Pyrosequencing and Quantitative Real-Time PCR to Analyze Complex Microbial Communities. In: Kwon, Y., Ricke, S. (eds) High-Throughput Next Generation Sequencing. Methods in Molecular Biology, vol 733. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-089-8_8

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  • DOI: https://doi.org/10.1007/978-1-61779-089-8_8

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-088-1

  • Online ISBN: 978-1-61779-089-8

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