Jump to ContentJump to Main Navigation
Show Summary Details
More options …

Open Life Sciences

formerly Central European Journal of Biology

Editor-in-Chief: Ratajczak, Mariusz

1 Issue per year


IMPACT FACTOR 2016 (Open Life Sciences): 0.448

CiteScore 2016: 1.02

SCImago Journal Rank (SJR) 2016: 0.329
Source Normalized Impact per Paper (SNIP) 2016: 0.621

Open Access
Online
ISSN
2391-5412
See all formats and pricing
More options …
Volume 8, Issue 5

Issues

A practical introduction to microbial community sequencing

Dominika Chmolowska
Published Online: 2013-03-07 | DOI: https://doi.org/10.2478/s11535-013-0155-8

Abstract

The use of molecular methods is gaining popularity throughout the field of microbial community ecology studies thanks to their flexibility of application, which ranges from community structure to function and trait determination. Nonetheless, there are environmental microbiologists, who are new in the field and are just starting to get to grips with the genetic tool box. It is for them that this practitioner’s mini-review was compiled. The methods available for microbial community structure analysis are discussed, after which, the reader is introduced to sequencing, as this tool is the most appropriate and has seen the greatest development in recent years. A focus on the practical aspects of the methodology is maintained throughout. The sample preparation procedure from extraction to sequencing is described. Different applications and considerations of sequencing are briefly explained, including clone library sequencing vs. amplicon library sequencing, shotgun-metagenomics vs. metatranscriptomics and the ‘double RNA approach’.

Keywords: Microbial community structure; Metagenomics

  • [1] Janssen P.H., Yates P.S., Grinton B.E., Taylor P.M., Sait M., Improved Culturability of Soil Bacteria and Isolation in Pure Culture of Novel Members of the Divisions Acidobacteria, Actinobacteria, Proteobacteria, and Verrucomicrobia, Appl. Environ. Microbiol., 2002, 68,5, 2391–2396 http://dx.doi.org/10.1128/AEM.68.5.2391-2396.2002CrossrefGoogle Scholar

  • [2] Nichols D., Cahoon N., Trakhtenberg E.M., Pham L., Mehta A, Belanger A., et al., Use of ichip for high-throughput in situ cultivation of “uncultivable” microbial species, Appl. Environ. Microbiol., 2010, 76, 2445–2450 http://dx.doi.org/10.1128/AEM.01754-09CrossrefGoogle Scholar

  • [3] Stefanowicz A., The Biolog Plates Technique as a Tool in Ecological Studies of Microbial Communities, Polish. Jour. Environ. Stud., 2006, 15, 669–676 Google Scholar

  • [4] Pinkart H.C., Ringelberg D.B., Piceno Y.M., MacNaughton S.J., White D.C., Biochemical Approaches to Biomass Measurements and Community Structure Analysis, In: Hurst C.J., Crawford R.L., Knudsen G.R., McInerney M.J., Stetzenbach L.D. (Eds.), Manual of Environmental Microbiology, ASM Press, Washington D.C., 2002 Google Scholar

  • [5] Olsson P., Signature fatty acids provide tools for determination of the distribution and interactions of mycorrhizal fungi in soil, FEMS Microbiol. Ecol., 1999, 29, 303–310 http://dx.doi.org/10.1111/j.1574-6941.1999.tb00621.xCrossrefGoogle Scholar

  • [6] Frostegård Å., Tunlid A., Bååth E., Use and misuse of PLFA measurements in soils, Soil Biol. Biochem., 2011, 43, 1621–1625 http://dx.doi.org/10.1016/j.soilbio.2010.11.021CrossrefGoogle Scholar

  • [7] Tyson G.W., Chapman J, Hugenholtz P, Allen E.E., Ram R.J., Richardson P.M., et al., Community structure and metabolism through reconstruction of microbial genomes from the environment, Nature, 2004, 428, 37–43 http://dx.doi.org/10.1038/nature02340CrossrefGoogle Scholar

  • [8] Simon C., Daniel R., Metagenomic analyses: past and future trends, Appl. Environ. Microbiol., 2011, 77, 1153–1161 http://dx.doi.org/10.1128/AEM.02345-10CrossrefGoogle Scholar

  • [9] Cantarel B.L., Erickson A.R., VerBerkmoes N.C., Erickson B.K., Carey P.A, Pan C., et al., Strategies for metagenomic-guided whole-community proteomics of complex microbial environments, PLoS One, 2011, 6, e27173 http://dx.doi.org/10.1371/journal.pone.0027173CrossrefGoogle Scholar

  • [10] Huang W.E., Griffiths R.I., Thompson I.P., Bailey M.J., Whiteley A.S., Raman Microscopic Analysis of Single Microbial Cells, Anal. Chem., 2004, 76, 4452–4458 http://dx.doi.org/10.1021/ac049753kCrossrefGoogle Scholar

  • [11] Harz M., Rösch P., Popp J., Vibrational spectroscopy-a powerful tool for the rapid identification of microbial cells at the single-cell level, Cytometry, 2009, 75A, 104–113 http://dx.doi.org/10.1002/cyto.a.20682CrossrefGoogle Scholar

  • [12] Adamczyk J., Hesselsoe M., Iversen N., Horn M., Lehner A., Nielsen P.H., et al., The Isotope Array, a New Tool That Employs Substrate-Mediated Labeling of rRNA for Determination of Microbial Community Structure and Function, Appl. Environ. Microbiol., 2003, 69, 6875–6887 http://dx.doi.org/10.1128/AEM.69.11.6875-6887.2003CrossrefGoogle Scholar

  • [13] Okabe S., Kindaichi T., Ito T., MAR-FISH — An Ecophysiological Approach to Link Phylogenetic Affiliation and In Situ Metabolic Activity of Microorganisms at a Single-Cell Resolution, Microbes Environ., 2004, 19, 83–98 http://dx.doi.org/10.1264/jsme2.19.83CrossrefGoogle Scholar

  • [14] Musat N., Halm H., Winterholler B., Hoppe P., Peduzzi S., Hillion F., et al., A single-cell view on the ecophysiology of anaerobic phototrophic bacteria, Proc Natl Acad Sci USA, 2008, 105, 17861–17866 http://dx.doi.org/10.1073/pnas.0809329105CrossrefGoogle Scholar

  • [15] Schippers A., Neretin L.N., Quantification of microbial communities in near-surface and deeply buried marine sediments on the Peru continental margin using realtime PCR, Environ. Microbiol., 2006, 8, 1251–1260 http://dx.doi.org/10.1111/j.1462-2920.2006.01019.xCrossrefGoogle Scholar

  • [16] DeSantis T.Z., Brodie E.L., Moberg J.P., Zubieta I.X., Piceno Y.M., Andersen G.L., High-density universal 16S rRNA microarray analysis reveals broader diversity than typical clone library when sampling the environment, Microbial Ecol., 2007, 53, 371–383 http://dx.doi.org/10.1007/s00248-006-9134-9CrossrefGoogle Scholar

  • [17] He Z., Gentry T.J., Schadt C.W., Wu L., Liebich J., Chong S.C., et al., GeoChip: a comprehensive microarray for investigating biogeochemical, ecological and environmental processes, The ISME Journal, 2007, 1, 67–77 http://dx.doi.org/10.1038/ismej.2007.2CrossrefGoogle Scholar

  • [18] Wilson K.H., Wilson W.J., Jennifer L., Desantis T.Z., Viswanathan V.S., Kuczmarski T.A., et al., High-Density Microarray of Small-Subunit Ribosomal DNA Probes High-Density Microarray of Small-Subunit Ribosomal DNA Probes, Appl.Environ. Microbiol., 2002, 68, 2535–2541 http://dx.doi.org/10.1128/AEM.68.5.2535-2541.2002CrossrefGoogle Scholar

  • [19] Hazen T.C., Rocha A.M., Techtmann S.M., Advances in monitoring environmental microbes, Cur. Opin. Biotech., 2012, 24, 1–8 Google Scholar

  • [20] Zwart G., van Hannen E.J., Kamst-van Agterveld M.P., van der Gucht K., Lindström E.S., van Wichelen J., et al., Rapid screening for freshwater bacterial groups by using reverse line blot hybridization, Appl. Environ. Microbiol., 2003, 69, 5875–5883 http://dx.doi.org/10.1128/AEM.69.10.5875-5883.2003CrossrefGoogle Scholar

  • [21] Nocker A., Burr M., Camper A.K., Genotypic Microbial Community Profiling: A Critical Technical Review, Microb. Ecol., 2007, 54, 276–289 http://dx.doi.org/10.1007/s00248-006-9199-5CrossrefGoogle Scholar

  • [22] Schütte U.M.E., Abdo Z., Bent S.J., Shyu C., Williams C.J., Pierson J.D., Forney L.J., Advances in the use of terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes to characterize microbial communities, Appl. Microbiol. Biotechnol., 2008, 80, 365–380 http://dx.doi.org/10.1007/s00253-008-1565-4CrossrefGoogle Scholar

  • [23] Prosser J.I., Replicate or lie, Environ. Microbiol., 2010, 12, 1806–1810 http://dx.doi.org/10.1111/j.1462-2920.2010.02201.xCrossrefGoogle Scholar

  • [24] Magurran E.E., McGill B.J., Biological diversity: frontiers in measurement and assessment, Oxford University Press, Oxford, 2011 Google Scholar

  • [25] Delmont T.O., Robe P., Cecillon S., Clark I.M., Constancias F., Simonet P., et al., Accessing the Soil Metagenome for Studies of Microbial Diversity, Appl. Env. Microbiol., 2011, 77, 1315–1324 http://dx.doi.org/10.1128/AEM.01526-10CrossrefGoogle Scholar

  • [26] Ovreas L., Curtis T.P., Microbial diversity and ecology, In: Magurran E.E., McGill B.J. (Eds.), Biological diversity: frontiers in measurement and assessment, Oxford University Press, Oxford, 2011 Google Scholar

  • [27] Simister R.L., Schmitt S., Taylor M.W., Evaluating methods for the preservation and extraction of DNA and RNA for analysis of microbial communities in marine sponges, J. Exp. Mar. Biol. Ecol., 2011, 397, 38–43 http://dx.doi.org/10.1016/j.jembe.2010.11.004CrossrefGoogle Scholar

  • [28] Masek T., Vopalensky V., Suchomelova P., Pospisek M., Denaturing RNA electrophoresis in TAE agarose gels, Anal. Biochem., 2005, 336, 46–50 http://dx.doi.org/10.1016/j.ab.2004.09.010CrossrefGoogle Scholar

  • [29] Blagodatskaya E.V., Blagodatskiı S.A., Anderson T.H., Quantitative isolation of microbial DNA from the different types of soils of natural and agricultural ecosystems, Microbiology, 2003, 72, 840–846 Google Scholar

  • [30] Aoshima H., Kimura A., Shibutan A., Okada C., Matsumiya Y., Kubo M., Evaluation of soil bacterial biomass using environmental DNA extracted by slow-stirring method, Appl. Microbiol. Biotech., 2006, 71, 875–880 http://dx.doi.org/10.1007/s00253-005-0245-xCrossrefGoogle Scholar

  • [31] Wang Y., Qian P.-Y., Conservative Fragments in Bacterial 16S rRNA Genes and Primer Design for 16S Ribosomal DNA Amplicons in Metagenomic Studies, PLoS One, 2009, 4, e7401 http://dx.doi.org/10.1371/journal.pone.0007401CrossrefGoogle Scholar

  • [32] Klindworth A., Pruesse E., Schweer T., Peplies J., Quast C., Horn M., et al., Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies, Nucl. Acid Res., 2012, 1–11 Google Scholar

  • [33] Mao D-P., Zhou Q., Chen Ch-Y., Quan Z-X., Coverage evaluation of universal bacterial primers using the metagenomic datasets, BMC Microbiology, 2012, 12, 66 http://dx.doi.org/10.1186/1471-2180-12-66CrossrefGoogle Scholar

  • [34] Walsh P.S., Erlich H.A., Higuchi R., Preferential PCR Amplification of Alleles: Mechanisms and Solutions, Genome Res., 1992, 1, 241–250 http://dx.doi.org/10.1101/gr.1.4.241CrossrefGoogle Scholar

  • [35] Abd-Elsalam K.A., Bioinformatic tools and guideline for PCR primer design, Afr. Jour. Biotech., 2003, 2, 91–95 Google Scholar

  • [36] Chen Z., Zhang Y., Dimethyl sulfoxide targets phage RNA polymerases to promote transcription, Biochem. Biophys. Res. Comm., 2005, 333, 664–670 http://dx.doi.org/10.1016/j.bbrc.2005.05.166CrossrefGoogle Scholar

  • [37] Rapley R., Enhancing PCR Amplification and Sequencing Using DNA-Binding Proteins, Mol. Biotech., 1994, 2, 295–298 http://dx.doi.org/10.1007/BF02745882CrossrefGoogle Scholar

  • [38] Sipos R., Székely A.J., Palatinszky M., Révész S., Márialigeti K., Nikolausz M., Effect of primer mismatch, annealing temperature and PCR cycle number on 16S rRNA gene-targetting bacterial community analysis, FEMS Microbiol. Ecol., 2007, 60, 341–350 http://dx.doi.org/10.1111/j.1574-6941.2007.00283.xCrossrefGoogle Scholar

  • [39] Mardis E.R., A decade’s perspective on DNA sequencing technology, Nature, 2011, 470, 198–203 http://dx.doi.org/10.1038/nature09796CrossrefGoogle Scholar

  • [40] Glenn T.C., Field guide to next-generation DNA sequencers, Mol. Ecol. Res., 2011, 11, 759–769 http://dx.doi.org/10.1111/j.1755-0998.2011.03024.xCrossrefGoogle Scholar

  • [41] Shokralla S., Spall J.L., Gibson J.F., Hajibabaei M., Next-generation sequencing technologies for environmental DNA research, Molecular Ecology, 2012, 21, 1794–1805 http://dx.doi.org/10.1111/j.1365-294X.2012.05538.xCrossrefGoogle Scholar

  • [42] Ahmadian A., Ehn M., Hober S., Pyrosequencing: history, biochemistry and future, Clin. chim. acta, 2006, 363, 83–94 http://dx.doi.org/10.1016/j.cccn.2005.04.038CrossrefGoogle Scholar

  • [43] Margulies M., Egholm M., Altman W., Attiya S., Bader J.S., Bemben L.A., et al., Genome sequencing in microfabricated high-density picolitre reactors, Nature, 2005, 437, 376–380 Google Scholar

  • [44] Ronaghi M., Karamohamed S., Pettersson B., Uhlén M., Nyrén P., Real-time DNA sequencing using detection of pyrophosphate release, Anal. Biochem., 1996, 242, 84–89 http://dx.doi.org/10.1006/abio.1996.0432CrossrefGoogle Scholar

  • [45] Hamady M., Walker J.J., Harris J.K., Gold N.J., Knight R., Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex, Nat Methods, 2008, 5, 235–237 http://dx.doi.org/10.1038/nmeth.1184CrossrefGoogle Scholar

  • [46] Kunin V., Copeland A., Lapidus A., Mavromatis K., Hugenholtz P., A bioinformatician’s guide to metagenomics, Microbiol. Mol. Biol. Rev., 2008, 72, 557–578 http://dx.doi.org/10.1128/MMBR.00009-08CrossrefGoogle Scholar

  • [47] Wooley J.C., Godzik A., Friedberg I., A Primer on Metagenomics, PLoS Comput Biol, 2010, 6, e1000667 http://dx.doi.org/10.1371/journal.pcbi.1000667CrossrefGoogle Scholar

  • [48] Moran M.A., Metatranscriptomics: Eavesdropping on Complex Microbial Communities, Microbe, 2009, 4, 329–335 Google Scholar

  • [49] Urich T., Lanzén A., Qi J., Huson, D.H., Schleper C., Schuster S.C., Simultaneous assessment of soil microbial community structure and function through analysis of the meta-transcriptome, PLoS ONE, 2008, 3, e2527 http://dx.doi.org/10.1371/journal.pone.0002527CrossrefGoogle Scholar

  • [50] Lanzén A., Jørgensen S.L., Bengtsson M.M., Jonassen I., Ovreas L., Urich T., Exploring the composition and diversity of microbial communities at the Jan Mayen hydrothermal vent field using RNA and DNA, FEMS Microbiol. Ecol., 2011, 77, 577–589 http://dx.doi.org/10.1111/j.1574-6941.2011.01138.xCrossrefGoogle Scholar

  • [51] Gilbert M.T., Tomsho L.P., Rendulic S., Packard M., Drautz D.I., Sher A., et al., Whole-genome shotgun sequencing of mitochondria from ancient hair shafts, Science, 2007, 317, 1927–1930 http://dx.doi.org/10.1126/science.1146971CrossrefGoogle Scholar

About the article

Published Online: 2013-03-07

Published in Print: 2013-03-01


Citation Information: Open Life Sciences, Volume 8, Issue 5, Pages 399–409, ISSN (Online) 2391-5412, DOI: https://doi.org/10.2478/s11535-013-0155-8.

Export Citation

© 2013 Versita Warsaw. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License. BY-NC-ND 3.0

Comments (0)

Please log in or register to comment.
Log in