Using pyrosequencing and quantitative PCR to analyze microbial communities

doi:10.1007/s11783-011-0303-9

Front Envir Sci Eng Chin

2011, Vol. 5

Issue (1) : 21-27 https://doi.org/10.1007/s11783-011-0303-9

REVIEW ARTICLE

Using pyrosequencing and quantitative PCR to analyze microbial communities

Husen ZHANG(

)

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

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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.

Keywords polymerase chain reaction (PCR) microbial communities pyrosequencing gut microbial fuel cell sludge

Corresponding Author(s): ZHANG Husen,Email:huzhang@mail.ucf.edu

Issue Date: 05 March 2011

Cite this article:

Husen ZHANG. Using pyrosequencing and quantitative PCR to analyze microbial communities[J]. Front Envir Sci Eng Chin, 2011, 5(1): 21-27.

URL:

https://academic.hep.com.cn/fese/EN/10.1007/s11783-011-0303-9
https://academic.hep.com.cn/fese/EN/Y2011/V5/I1/21

Fig.1 Schematic drawing of the 454 pyrosequencing work flow, adapted from Gharizadeh et al. [] and Margulies et al. []

Fig.2 QPCR fluorescent dye chemistries. A, SYBR Green I. During amplification, SYBR Green I binds newly synthesized double-stranded DNA and yields an increase in fluorescence. Unbound dyes do not fluoresce. B, TaqMan? probe. An oligonucleotide probe labeled with a 5′ reporter fluorophore and 3′ quencher binds target DNA during primer annealing. During primer extension, polymerase partially displaces the probe and cleaves the reporter, and the unquenched reporter fluoresces

Fig.3 Using pyrosequencing (P) and QPCR (Q) in parallel to identify anode biofilm community members in an MEC. The black arrows indicate electron flow path when methanogenesis was allowed, and the green arrows indicate the flow when methanogenesis was inhibited. The cross sign (red) indicates the pathways that were not observed. The method used to identify key members mediating the observed electron flow is indicated as either P or Q. For example, ethanol fermenters were identified by using P (pyrosequencing), and hydrogenotrophic methanogens were identified with Q (QPCR)

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