Rapid quantitative profiling of complex microbial populations

Department of Biochemistry, Stanford University, Palo Alto, California, United States
Nucleic Acids Research (Impact Factor: 9.11). 02/2006; 34(1):e5. DOI: 10.1093/nar/gnj007
Source: PubMed


Diverse and complex microbial ecosystems are found in virtually every environment on earth, yet we know very little about their composition and ecology. Comprehensive identification and quantification of the constituents of these microbial communities--a 'census'--is an essential foundation for understanding their biology. To address this problem, we developed, tested and optimized a DNA oligonucleotide microarray composed of 10,462 small subunit (SSU) ribosomal DNA (rDNA) probes (7167 unique sequences) selected to provide quantitative information on the taxonomic composition of diverse microbial populations. Using our optimized experimental approach, this microarray enabled detection and quantification of individual bacterial species present at fractional abundances of <0.1% in complex synthetic mixtures. The estimates of bacterial species abundance obtained using this microarray are similar to those obtained by phylogenetic analysis of SSU rDNA sequences from the same samples--the current 'gold standard' method for profiling microbial communities. Furthermore, probes designed to represent higher order taxonomic groups of bacterial species reliably detected microbes for which there were no species-specific probes. This simple, rapid microarray procedure can be used to explore and systematically characterize complex microbial communities, such as those found within the human body.

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    • "To rapidly characterize the members of microbial communities present in complex environments, numerous phylogenetic oligonucleotide arrays (POAs) have been developed using the SSU rRNA biomarker (Loy et al., 2002; Wilson et al., 2002; Brodie et al., 2006, 2007; Palmer et al., 2006; DeSantis et al., 2007; Hazen et al., 2010). Fully automated software and manual approaches have both been developed to design POAs (Tables 2.1–2.3). "
    Microarrays: Current Technology, Innovations and Applications, Edited by He, Zhili, 08/2014: pages 250; Academic Press.
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    • "As the feeding behaviour of fish might undergo spatio-temporal changes and the relative frequency between different prey species would be quite variable, quantification would be desirable; however, this is not possible at the current stage of INV-CHIP development. Methods already used to quantify microbial populations [75] can be adapted to marine organisms to quantitatively assess the diverse composition of natural populations. However, the INV-CHIP can be used for regular monitoring of the gut content for ecosystem functioning analysis and to investigate the common prey of commercially-relevant fishery resources. "
    Microarrays: Principles, Applications and Technologies, Edited by Rogers, J. V., 01/2014: pages 101-134; Nova Science Publishers, Inc.., ISBN: 9781629486697
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    • "To amplify the V1/V2 16S rRNA gene regions [17], a 30ng of purified DNA was amplified with a TOPsimpleTM DryMIX solution (Enzynomics, Daejeon, Korea) were amplified with the primer pair 8F (5’-AGAGTTTGAT CCTGGCTCAG-3’) and 338R (5’-TGCTGCCTCC CGTAGGAGT-3’) containing eight base sample-specific barcode sequences (Table S2) and common linker (TC for forward and CA for reverse primer) sequences at the 5’ end [18]. This approach allowed the analysis of PCR products from multiple samples in parallel on a single 454 picotiter plate, and the ability to re-sort the sequences into order [19]. Thermocycling was conducted in a C 1000 Thermal Cycler (Bio-Rad, Hercules, CA, USA) under the following conditions: initial denaturation at 94 °C for 2 minutes; 18 cycles of denaturation at 94 °C for 30 seconds, annealing at 55 °C for 30 seconds, extension at 72 °C for 1 minute, and a final extension at 72 °C for 10 minutes. "
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