DNA-, RNA-, and Protein-Based Stable-Isotope Probing for High-Throughput Biomarker Analysis of Active Microorganisms

Allbwn ymchwil: Pennod mewn Llyfr/Adroddiad/Trafodion CynhadleddPennodadolygiad gan gymheiriaid

Fersiynau electronig

Dangosydd eitem ddigidol (DOI)

  • Eleanor Jameson
    The University of Warwick
  • Martin Taubert
    Friedrich Schiller University, Jena
  • Sara Coyotzi
    Waterloo University
  • Yin Chen
    The University of Warwick
  • Özge Eyice
    Queen Mary, University of London
  • Hendrik Schäfer
    The University of Warwick
  • J Colin Murrell
    The University of East Anglia
  • Josh D Neufeld
    Waterloo University
  • Marc G Dumont
    Southampton University

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 omics-based analyses.

Allweddeiriau

Iaith wreiddiolSaesneg
TeitlMetagenomics: Methods and Protocols: 1539 (Methods in Molecular Biology)
Man cyhoeddiNew York, NY
CyhoeddwrHumana Press
Tudalennau57-74
Nifer y tudalennau18
Cyfrol1539
Argraffiad2nd
ISBN (Electronig)9781493966912
ISBN (Argraffiad)9781493966899
Dynodwyr Gwrthrych Digidol (DOIs)
StatwsCyhoeddwyd - 30 Tach 2016
Cyhoeddwyd yn allanolIe

Cyfres gyhoeddiadau

EnwMethods in Molecular Biology
CyhoeddwrHumana Press
ISSN (Argraffiad)1064-3745
Gweld graff cysylltiadau