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RETRACTED ARTICLE: Using pyrosequencing and quantitative PCR to analyze microbial communities

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An Erratum to this article was published on 03 June 2011

An Erratum to this article was published on 03 June 2011

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 polymerase chain reaction (QPCR) allows quantitative monitoring of specific community members over time, space, or different environmental conditions. In this review, the principles of these two methods and their complementary applications in studying microbial ecology in bioenvironmental systems are discussed. The parallel sequencing of amplicon libraries and using barcodes to differentiate multiple samples in a pyrosequencing run are explained. The best procedures and chemistries for QPCR amplifications are also described and advantages of applying automation to increase accuracy are addressed. Three examples in which pyrosequencing and QPCR were used together to define and quantify members of microbial communities are provided: 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. The key findings in these systems and how both methods were used in concert to achieve those findings are highlighted.

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References

  1. Rittmann B E, Hausner M, Loffler F, Love N G, Muyzer G, Okabe S, Oerther D B, Peccia J, Raskin L, Wagner M. A vista for microbial ecology and environmental biotechnology. Environmental Science & Technology, 2006, 40(4): 1096–1103

    Article  Google Scholar 

  2. Rittmann B E, Krajmalnik-Brown R, Halden R U. Pre-genomic, genomic and post-genomic study of microbial communities involved in bioenergy. Nature Reviews Microbiology, 2008, 6(8): 604–612

    Article  CAS  Google Scholar 

  3. Coates J D, Michaelidou U, Bruce R A, O’Connor S M, Crespi J N, Achenbach L A. Ubiquity and diversity of dissimilatory (per) chlorate-reducing bacteria. Applied and Environmental Microbiology, 1999, 65: 5234–5241

    CAS  Google Scholar 

  4. Wu J, Unz R F, Zhang H, Logan B E. Persistence of perchlorate and the relative numbers of perchlorate- and chlorate-respiring microorganisms in natural waters, soils, and wastewater. Bioremediation Journal, 2001, 5(2): 119–130

    Article  CAS  Google Scholar 

  5. Hugenholtz P, Goebel B M, Pace N R. Impact of cultureindependent studies on the emerging phylogenetic view of bacterial diversity. Journal of Bacteriology, 1998, 180: 4765–4774

    CAS  Google Scholar 

  6. Amann R I, Ludwig W, Schleifer K H. Phylogenetic identification and in-situ detection of individual microbial cells without cultivation. Microbiological Reviews, 1995, 59: 143–169

    CAS  Google Scholar 

  7. Zhang H, DiBaise J K, Zuccolo A, Kudrna D, Braidotti M, Yu Y, Parameswaran P, Crowell MD, Wing R, Rittmann B E, Krajmalnik-Brown R. Human gut microbiota in obesity and after gastric-bypass. Proceedings of the National Academy of Sciences of the United States of America, 2009, 106(7): 2365–2370

    Article  CAS  Google Scholar 

  8. Eckburg P B, Bik E M, Bernstein C N, Purdom E, Dethlefsen L, Sargent M, Gill S R, Nelson K E, Relman D A. Diversity of the human intestinal microbial flora. Science, 2005, 308(5728): 1635–1638

    Article  Google Scholar 

  9. Hugenholtz P, Tyson G W. Microbiology — Metagenomics. Nature, 2008, 455(7212): 481–483

    Article  CAS  Google Scholar 

  10. Jones R T, Robeson M S, Lauber C L, Hamady M, Knight R, Fierer N. A comprehensive survey of soil acidobacterial diversity using pyrosequencing and clone library analyses. ISME Journal, 2009, 3(4): 442–453

    Article  CAS  Google Scholar 

  11. Hamady M, Walker J J, Harris J K, Gold N J, Knight R. Errorcorrecting barcoded primers for pyrosequencing hundreds of samples in multiplex. Nature Methods, 2008, 5(3): 235–237

    Article  CAS  Google Scholar 

  12. Suzuki M T, Taylor L T, DeLong E F. Quantitative analysis of small-subunit rRNA genes in mixed microbial populations via 5′-nuclease assays. Applied and Environmental Microbiology, 2000, 66(11): 4605–4614

    Article  CAS  Google Scholar 

  13. Yu Y, Lee C, Kim J, Hwang S. Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction. Biotechnology and Bioengineering, 2005, 89(6): 670–679

    Article  CAS  Google Scholar 

  14. Ritalahti K M, Amos B K, Sung Y, Wu Q, Koenigsberg S S, Loffler F E. Quantitative PCR targeting 16S rRNA and reductive dehalogenase genes simultaneously monitors multiple Dehalococcoides strains. Applied and Environmental Microbiology, 2006, 72(4): 2765–2774

    Article  CAS  Google Scholar 

  15. Margulies M, Egholm M, Altman, W E, Attiya S, Bader J S, Bemben L A, Berka J, Braverman MS, Chen Y J, Chen Z T, Dewell S B, Du L, Fierro JM, Gomes X V, Godwin B C, He W, Helgesen S, Ho C H, Irzyk G P, Jando S C, Alenquer M L I, Jarvie T P, Jirage K B, Kim J B, Knight J R, Lanza J R, Leamon J H, Lefkowitz SM, Lei M, Li J, Lohman K L, Lu H, Makhijani V B, McDade K E, McKenna MP, Myers EW, Nickerson E, Nobile J R, Plant R, Puc B P, Ronan M T, Roth G T, Sarkis G J, Simons J F, Simpson J W, Srinivasan M, Tartaro K R, Tomasz A, Vogt K A, Volkmer G A, Wang S H, Wang Y, Weiner M P, Yu P G, Begley R F, Rothberg J M. Genome sequencing in microfabricated high-density picolitre reactors. Nature, 2005, 437: 376–380

    CAS  Google Scholar 

  16. Gharizadeh B, Kalantari M, Garcia C A, Johansson B, Nyren P. Typing of human papillomavirus by pyrosequencing. Laboratory Investigation, 2001, 81(5): 673–679

    Article  CAS  Google Scholar 

  17. Zhang T, Fang H H. Applications of real-time polymerase chain reaction for quantification of microorganisms in environmental samples. Applied Microbiology and Biotechnology, 2006, 70(3): 281–289

    Article  CAS  Google Scholar 

  18. Talbot G, Topp E, Palin M F, Masse D I. Evaluation of molecular methods used for establishing the interactions and functions of microorganisms in anaerobic bioreactors. Water Research, 2008, 42(3): 513–537

    Article  CAS  Google Scholar 

  19. Sogin M L, Morrison H G, Huber J A, Mark Welch D, Huse S M, Neal P R, Arrieta J M, Herndl G J. Microbial diversity in the deep sea and the underexplored “rare biosphere”. In: Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(32): 12115–12120

    Article  CAS  Google Scholar 

  20. Rittmann B E, Lee H, Zhang H, Alder J, Banazak J E, Lopez R. Fullscale application of Focused-Pulsed pre-treatment for improving biosolids digestion and conversion to methane. Water Science and Technology, 2008, 58(10): 1895–1901

    Article  CAS  Google Scholar 

  21. Zhang H, Banaszak J E, Parameswaran P, Alder J, Krajmalnik-Brown R, Rittmann B E. Focused-Pulsed sludge pre-treatment increases the bacterial diversity and relative abundance of acetoclastic methanogens in a full-scale anaerobic digester. Water Research, 2009, 43(18): 4517–4526

    Article  CAS  Google Scholar 

  22. Parameswaran P, Zhang H, Torres C I, Rittmann B E, Krajmalnik-Brown R. Microbial community structure in a biofilm anode fed with a fermentable substrate: The significance of hydrogen scavengers. Biotechnology and Bioengineering, 2010, 105(1): 69–78

    Article  CAS  Google Scholar 

  23. Parameswaran P, Torres C I, Lee H S, Krajmalnik-Brown R, Rittmann B E. Syntrophic interactions among anode respiring bacteria (ARB) and non-ARB in a biofilm anode: electron balances. Biotechnology and Bioengineering, 2009, 103(3): 513–523

    Article  CAS  Google Scholar 

  24. Liu H, Cheng S, Logan B E. Production of electricity from acetate or butyrate using a single-chamber microbial fuel cell. Environmental Science & Technology, 2005, 39(2): 658–662

    Article  CAS  Google Scholar 

  25. Jung S, Regan JM. Comparison of anode bacterial communities and performance in microbial fuel cells with different electron donors. Applied Microbiology and Biotechnology, 2007, 77(2): 393–402

    Article  CAS  Google Scholar 

  26. Torres C I, Marcus A K, Parameswaran P, Rittmann B E. Kinetic experiments for evaluating the Nernst-Monod model for anode-respiring bacteria (ARB) in a biofilm anode. Environmental Science & Technology, 2008, 42(17): 6593–6597

    Article  CAS  Google Scholar 

  27. Lee H S, Parameswaran P, Kato-Marcus A, Torres C I, Rittmann B E. Evaluation of energy-conversion efficiencies in microbial fuel cells (MFCs) utilizing fermentable and non-fermentable substrates. Water Research, 2008, 42(6–7): 1501–1510

    Article  CAS  Google Scholar 

  28. Logan B E. Exoelectrogenic bacteria that power microbial fuel cells. Nature Reviews Microbiology, 2009, 7(5): 375–381

    Article  CAS  Google Scholar 

  29. Kunin V, Engelbrektson A, Ochman H, Hugenholtz P. Wrinkles in the rare biosphere: pyrosequencing errors can lead to artificial inflation of diversity estimates. Environmental Microbiology, 2010, 12(1): 118–123

    Article  CAS  Google Scholar 

  30. Quince C, Lanzen A, Curtis T P, Davenport R J, Hall N, Head I M, Read L F, Sloan WT. Accurate determination of microbial diversity from 454 pyrosequencing data. Nature Methods, 2009, 6(9): 639–641

    Article  CAS  Google Scholar 

  31. Engelbrektson A, Kunin V, Wrighton K C, Zvenigorodsky N, Chen F, Ochman H, Hugenholtz P. Experimental factors affecting PCR-based estimates of microbial species richness and evenness. ISME Journal, 2010, 4(5): 642–647

    Article  CAS  Google Scholar 

  32. Zhou J, Kang S, Schadt C W, Garten C T. Spatial scaling of functional gene diversity across various microbial taxa. In: Proceedings of the National Academy of Sciences of the United States of America, 2008, 105(22): 7768–7773

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Civil, Environmental, and Construction Engineering, University of Central Florida, Orlando, FL, 32816, USA

    Husen Zhang

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  1. Husen Zhang
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Correspondence to Husen Zhang.

Additional information

This article has been retracted because it already was published as “High-Throughput Next Generation Sequencing” in Methods in Molecular Biology, 2011, Volume 733, Part 3, 107-128.

An erratum to this article can be found at http://dx.doi.org/10.1007/s11783-011-0342-2

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Zhang, H. RETRACTED ARTICLE: Using pyrosequencing and quantitative PCR to analyze microbial communities. Front. Environ. Sci. Eng. China 5, 21–27 (2011). https://doi.org/10.1007/s11783-011-0303-9

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  • DOI: https://doi.org/10.1007/s11783-011-0303-9

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