SOIL METAGENOMIC: WHERE IS THE DIVERSITY?
Résumé
A novel strategy for recovering microbial DNA diversity has been developed. Less than 1% of soil microbial diversity is considered to be cultivable by traditional techniques. To overcome this problem, metagenomic approaches appeared, using direct DNA extraction from soil. The proposed methods are widely used although they have numerous biases that limit the quantity of metagenomic DNA. In this study, we used different DNA separation techniques coupled with biodiversity analyses (microarray, cloning and sequencing of 16S rRNA genes (rrs), and ribosomal intergenic spacer analysis). We defined a strategy to increase soil extracted nucleic acid diversity. We applied three DNA separation techniques which significantly separate DNA diversity. These three techniques are based respectively on the cell density (Nycodenz density gradient gels), cell membrane resistance to lysis (cell lysis conditions), and DNA degradation (pulsed field electrophoresis gels). Although these respective methods are not without some overlap, we have shown that they can be adjusted to increase the relative diversity of the final DNA pool (based on phylogenetic analyses as mentioned above). In other words, by varying the conditions of the three methods and applying phylogenetic methods to track relative diversity and less represented species, the final DNA pool can be optimized for increased nucleic acid diversity. This strategy was compared to other more common approaches (including individual application of one of the three methods used here) in order to illustrate the advantages of this approach. This strategy produced increase diversity as measured by phylochips, RISA, and cloning and sequencing. In addition, this strategy can also be coupled to targeted DNA sizes for further use in clone libraries.