Environmental Metagenomics: Techniques

The requirements for accessing a larger proportion of ecosystem microorganisms than those typically "extracted" by isolation or enrichment techniques has led to the development and use of a range of metagenomic techniques. These techniques improve the accessibility of both functional and structural information concerning complex microbial communities. Some of this methodology development is described below.  Applications of these techniques are found throughout the other research sections (for example, metagenomic searching for PKS genes, or the use of metagenomic DNA for gene synthesis, or the screening of soil bacterial metagenomes for plant transgenes…)

DNA Extraction and Purification:

Our work with DNA extraction has two main objectives: 1) Recovery of the representative DNA from environmental samples and 2) Damage minimization in order to access relative long unbroken environmental DNA sequences.

Clone Libraries: Metasoil metaexplore

Our clone libraries usually contain either plasmid or cosmids.  In general, DNA fragments of a few kb are cloned in the plasmids and fragments of about 30 kb in the cosmids.
  
Screening Techniques:

Several different screening techniques have been applied including phenotypic (selection or election on Petri plates) and genotypic (with the hybridization of clone libraries densely spotted on membranes (order of 30 000 clones per membrane)

Microarrays:

Phylogenetic microarrays containing probes at a range of taxonomic levels are used in order to access the prokaryotic diversity in environmental samples.

Method and review articles (except for microarray work):

Demanèche, S., Philippot, L., David, M.M., Navarro, E., Vogel, T.M., Simonet, P. 2009. Characterization  of Denitrification Gene Clusters of Soil Bacteria via a Metagenomic Approach. Appl. Environ. Microbiol.75:

Demanèche, S., Philippot, L., David, M.M., Navarro, E., Simonet, P., Vogel, T.M. 2009. Evaluation of functional gene enrichment in a soil metagenomic clone library. J. Microbiol. Methods 74:

van Elsas, J.D., Costa, R., Jansson, J., Sjöling, S., Bailey, M., Nalin, R., Vogel, T.M. and van Overbeek, L. 2008. The metagenomics of disease suppressive soils – experiences from the METACONTROL project. Trends in Biotechnol. doi:10.1016/j.tibtech.2008.07.004

David M, Sapkota AR, Simonet P, Vogel, T. M. 2008. A novel and rapid  method for synthesizing positive controls or standards for  quantitative PCR. J. Microbiol. Methods73:73-77.

Lefevre F, Robe P, Jarrin C, Ginolhac A, Zago C, Auriol D, Vogel T.M.,  Simonet P and Nalin R. Drugs from hidden bugs: Discovery from untapped  resources. Res. Microbiol. 159:153-161.

Lombard, N., J. Bailly, R. Marmeisse and P. Simonet. 2006. La métagénomique des communautés microbiennes. Biofutur 268:24-27

Bertrand H., Poly F., Van T. V., Lombard N., Nalin R., Vogel T.M. and Simonet P. 2005. Improved quality of DNA extracted from soil suitable for metagenomic library construction. J. Microb. Methods 62 :1-11.

Robe, P., Nalin, R., Capellano, C., Vogel, T.M., and Simonet, P. 2003. Extraction of DNA from soil, European Journal of Soil Biology, 39:183-190.

Demanèche S., Jocteur-Monrozier L., Quiquampoix H. and Simonet P. 2001. Evaluation of Biological and Physical Protection against Nuclease Degradation of Clay-Bound Plasmid DNA. Appl. Environ. Microbiol. 2001. 67:293-299.

Courtois S., Frostegard A, Goransson P, Depret G, Jeannin P and Simonet P. 2001. Quantification of bacterial subgroups in soil; comparison of DNA extracted directly from soil or from cells previously released by density gradient centrifugation. Environ. Microbiol. 3 : 431-439.

Poly F., Chenu C., Simonet P., Rouiller J. and Jocteur Monrozier L. 2000. Differences between linear chromosomal and supercoiled plasmid DNA in their mechanisms and extent of adsorption on clay minerals. Langmuir. 16 : 1233-1238.

Frostegard A., Courtois S., Ramisse V., Clerc S ., Bernillon D., Le Gall F., Jeannin P., Nesme X. and Simonet P. 1999. Quantification of Bias Related to the Extraction of DNA Directly from Soils. Appl. Environ. Microbiol., 65 : 5409-5420.

Nalin R., Normand P., Simonet P. and Domenach A.M. 1999. Polymerase chain reaction and hybridization on DNA extracted from soil as a tool for Frankia spp. population distribution studies in soil. Can. J. Microbiol. 77 : 1239-1247.

Nesme X et Simonet P. 1997. Devenir de l'ADN dans le sol. OCL 4 (2):107-110.