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.

Original languageEnglish (US)
Pages (from-to)107-128
Number of pages22
JournalMethods in molecular biology (Clifton, N.J.)
Volume733
DOIs
StatePublished - 2011

Fingerprint

Real-Time Polymerase Chain Reaction
Automation
Libraries
Large Intestine
Biofilms
Sewage
Ecology
Automatic Data Processing
Electrodes
Technology
Polymerase Chain Reaction

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Integrating high-throughput pyrosequencing and quantitative real-time PCR to analyze complex microbial communities. / Zhang, Husen; Parameswaran, Prathap; Badalamenti, Jonathan; Rittmann, Bruce; Krajmalnik-Brown, Rosa.

In: Methods in molecular biology (Clifton, N.J.), Vol. 733, 2011, p. 107-128.

Research output: Contribution to journalArticle

@article{e40e724ed20e4ef38bbfd4e6757ccc52,
title = "Integrating high-throughput pyrosequencing and quantitative real-time PCR to analyze complex microbial communities.",
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.",
author = "Husen Zhang and Prathap Parameswaran and Jonathan Badalamenti and Bruce Rittmann and Rosa Krajmalnik-Brown",
year = "2011",
doi = "10.1007/978-1-61779-089-8_8",
language = "English (US)",
volume = "733",
pages = "107--128",
journal = "Methods in molecular biology (Clifton, N.J.)",
issn = "1064-3745",
publisher = "Humana Press",

}

TY - JOUR

T1 - Integrating high-throughput pyrosequencing and quantitative real-time PCR to analyze complex microbial communities.

AU - Zhang, Husen

AU - Parameswaran, Prathap

AU - Badalamenti, Jonathan

AU - Rittmann, Bruce

AU - Krajmalnik-Brown, Rosa

PY - 2011

Y1 - 2011

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=79960069580&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=79960069580&partnerID=8YFLogxK

U2 - 10.1007/978-1-61779-089-8_8

DO - 10.1007/978-1-61779-089-8_8

M3 - Article

C2 - 21431766

AN - SCOPUS:79960069580

VL - 733

SP - 107

EP - 128

JO - Methods in molecular biology (Clifton, N.J.)

JF - Methods in molecular biology (Clifton, N.J.)

SN - 1064-3745

ER -