Optimization of molecular methods and statistical procedures for forensic fingerprinting of microbial soil communities

Soil may constitute physical evidence that connects people to geographic locations. We studied several parameters of molecular fingerprinting of microbes from arid soils, revealing that (i) for extraction of soil DNA, the ‘total nucleic acid’ method provides higher spatial and temporal resolution than the PowerSoil® method; (ii) denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (TRFLP) were equally powerful at discriminating between soils; and (iii) the most effective restriction enzymes for TRFLP were MspI (for Archaea), HhaI (for Actinobacteria), AciI (for Fungi), TaqI (for Bacteria) and HaeIII (for α-proteobacteria). In addition, we optimized TRFLP data analysis procedures, including outlier and rare-terminal restriction fragments’ deletion, relativization and transformation. Overall, Bacterial fingerprints proved better than Actinobacteria and α-proteobacteria, providing higher spatial resolution (i.e. distinction between geographical sites) combined with lower temporal resolution (i.e. stable fingerprints regardless of sampling season).