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DNA-, RNA-, and protein-based stable-isotope probing for high-throughput biomarker analysis of active microorganisms

DNA-, RNA-, and protein-based stable-isotope probing for high-throughput biomarker analysis of active microorganisms
DNA-, RNA-, and protein-based stable-isotope probing for high-throughput biomarker analysis of active microorganisms
Stable-isotope probing (SIP) enables researchers to target active populations within complex microbial communities, which is achieved by providing growth substrates enriched in heavy isotopes, usually in the form of 13C, 18O, or 15N. After growth on the substrate and subsequent extraction of microbial biomarkers, typically nucleic acids or proteins, the SIP technique is used for the recovery and analysis of isotope-labeled biomarkers from active microbial populations. In the years following the initial development of DNA- and RNA-based SIP, it was common practice to characterize labeled populations by targeted gene analysis. Such approaches usually involved fingerprint-based analyses or sequencing of clone libraries containing 16S rRNA genes or functional marker gene amplicons. Although molecular fingerprinting remains a valuable approach for rapid confirmation of isotope labeling, recent advances in sequencing technology mean that it is possible to obtain affordable and comprehensive amplicon profiles, metagenomes, or metatranscriptomes from SIP experiments. Not only can the abundance of microbial groups be inferred from metagenomes, but researchers can bin, assemble, and explore individual genomes to build hypotheses about the metabolic capabilities of labeled microorganisms. Analysis of labeled mRNA is a more recent advance that can provide independent metatranscriptome-based analysis of active microorganisms. The power of metatranscriptomics is that mRNA abundance often correlates closely with the corresponding activity of encoded enzymes, thus providing insight into microbial metabolism at the time of sampling. Together, these advances have improved the sensitivity of SIP methods and allow the use of labeled substrates at ecologically relevant concentrations. Particularly as methods improve and costs continue to drop, we expect that the integration of SIP with multiple ‘omics-based methods will become prevalent components of microbial ecology studies, leading to further breakthroughs in our understanding of novel microbial populations and elucidation of the metabolic function of complex microbial communities. In this chapter we provide protocols for obtaining labeled DNA, RNA, and proteins that can be used for downstream ‘omic-based analyses.
1064-3745
57-74
Humana Press
Jameson, Eleanor
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Taubert, Martin
e4786f60-96d7-46bf-b36c-739d7aa6a9c6
Coyotzi, Sara
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Chen, Yin
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Eyice, Ozge
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Schaefer, Hendrik
eb1f6927-2112-4704-997b-5372bb5dab8e
Murrell, J. Colin
22f4dcaa-2fab-4b88-9365-0418bae53ec9
Neufeld, Josh D.
5e6989c6-ca4a-4311-a36d-3c2efbe08683
Dumont, Marc
afd9f08f-bdbb-4cee-b792-1a7f000ee511
Streit, Wolfgang
Daniel, Rolf
Jameson, Eleanor
00e2bf3d-789a-40f3-9af0-6450def09852
Taubert, Martin
e4786f60-96d7-46bf-b36c-739d7aa6a9c6
Coyotzi, Sara
ec7a460c-73d7-48d3-9b9b-a173dc6bc44e
Chen, Yin
32e9131d-b9f7-470e-96ba-e0586b9dde69
Eyice, Ozge
9060c46a-380e-4822-9a85-5ba7f20d97c9
Schaefer, Hendrik
eb1f6927-2112-4704-997b-5372bb5dab8e
Murrell, J. Colin
22f4dcaa-2fab-4b88-9365-0418bae53ec9
Neufeld, Josh D.
5e6989c6-ca4a-4311-a36d-3c2efbe08683
Dumont, Marc
afd9f08f-bdbb-4cee-b792-1a7f000ee511
Streit, Wolfgang
Daniel, Rolf

Jameson, Eleanor, Taubert, Martin, Coyotzi, Sara, Chen, Yin, Eyice, Ozge, Schaefer, Hendrik, Murrell, J. Colin, Neufeld, Josh D. and Dumont, Marc (2017) DNA-, RNA-, and protein-based stable-isotope probing for high-throughput biomarker analysis of active microorganisms. In, Streit, Wolfgang and Daniel, Rolf (eds.) Metagenomics: Methods and Protocols. (Methods in Molecular Biology, , (doi:10.1007/978-1-4939-6691-2_5), 1539) 2 ed. Humana Press, pp. 57-74. (doi:10.1007/978-1-4939-6691-2_5).

Record type: Book Section

Abstract

Stable-isotope probing (SIP) enables researchers to target active populations within complex microbial communities, which is achieved by providing growth substrates enriched in heavy isotopes, usually in the form of 13C, 18O, or 15N. After growth on the substrate and subsequent extraction of microbial biomarkers, typically nucleic acids or proteins, the SIP technique is used for the recovery and analysis of isotope-labeled biomarkers from active microbial populations. In the years following the initial development of DNA- and RNA-based SIP, it was common practice to characterize labeled populations by targeted gene analysis. Such approaches usually involved fingerprint-based analyses or sequencing of clone libraries containing 16S rRNA genes or functional marker gene amplicons. Although molecular fingerprinting remains a valuable approach for rapid confirmation of isotope labeling, recent advances in sequencing technology mean that it is possible to obtain affordable and comprehensive amplicon profiles, metagenomes, or metatranscriptomes from SIP experiments. Not only can the abundance of microbial groups be inferred from metagenomes, but researchers can bin, assemble, and explore individual genomes to build hypotheses about the metabolic capabilities of labeled microorganisms. Analysis of labeled mRNA is a more recent advance that can provide independent metatranscriptome-based analysis of active microorganisms. The power of metatranscriptomics is that mRNA abundance often correlates closely with the corresponding activity of encoded enzymes, thus providing insight into microbial metabolism at the time of sampling. Together, these advances have improved the sensitivity of SIP methods and allow the use of labeled substrates at ecologically relevant concentrations. Particularly as methods improve and costs continue to drop, we expect that the integration of SIP with multiple ‘omics-based methods will become prevalent components of microbial ecology studies, leading to further breakthroughs in our understanding of novel microbial populations and elucidation of the metabolic function of complex microbial communities. In this chapter we provide protocols for obtaining labeled DNA, RNA, and proteins that can be used for downstream ‘omic-based analyses.

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Accepted/In Press date: 16 February 2016
e-pub ahead of print date: 30 November 2016
Published date: 2017

Identifiers

Local EPrints ID: 416587
URI: http://eprints.soton.ac.uk/id/eprint/416587
ISSN: 1064-3745
PURE UUID: 0d005dcd-68e1-434b-8ece-e534f5c6fb6d
ORCID for Marc Dumont: ORCID iD orcid.org/0000-0002-7347-8668

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Date deposited: 03 Jan 2018 17:30
Last modified: 17 Dec 2019 01:33

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Contributors

Author: Eleanor Jameson
Author: Martin Taubert
Author: Sara Coyotzi
Author: Yin Chen
Author: Ozge Eyice
Author: Hendrik Schaefer
Author: J. Colin Murrell
Author: Josh D. Neufeld
Author: Marc Dumont ORCID iD
Editor: Wolfgang Streit
Editor: Rolf Daniel

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