Table 1: Advantages and limitations of the culture-independent methods usually used to investigate soil microbial communities.

MethodAdvantagesLimitations

SSCP (single-strand conformation polymorphism) [14]Separates amplified 16S ssDNA by sequence-dependent higher-order structure; great simplicity and speed; automation possible. No GC-clamp is necessary and no gradient gels.Formation of heteroduplexes; limited phylogenetic information; only short fragments (between 150–400 nucleotides) can be optimally separated. High rate of reannealing during electrophoresis; biases introduced by conformation variations.

DGGE (denaturing-gradient gel electrophoresis) [1517]Separates amplified molecules by %GC content on a denaturing gradient; excellent and effective to follow changes of microbial communities in time and space; well suited for monitoring complex communities dominated by a few members; allows phylogenetic identification through excision and sequencing of individual bands.Limited to dominant communities; samples with high levels of diversity are difficult to resolve; phylogenetic information is limited to bands that are able to be removed and sequenced. A single band does not always mean a single strain; needs careful calibration; limited to DNA fragments typically below 500 bp in size.

TGGE (temperature-gradient gel electrophoresis) [18]Separates amplified molecules by %GC content on a temperature gradient; operates on the same principle as DGGE; taxonomic information could be obtained from isolated bands.Provides approximately the same degree of specificity as DGGE and possesses the same advantages and limitations.

RISA (ribosomal RNA intergenic spacer analysis) [1921]Amplifies the prokaryotic ribosomal intergenic region creating a community profile based on the species-specific length polymorphisms in this region; great simplicity and speed; automation is possible.The preferential amplification of shorter sequences is a particular concern; biases imposed by secondary structures in the rDNA genes flanking the amplified region may also pose a problem; small database.

T-RFLP (terminal restriction fragment length polymorphism) [2225]Separates amplified and digested molecules according to their length; only terminal restriction fragments (TRFs) are detected and used for qualitative and quantitative analysis; powerful tool for assessing diversity and structure of complex microbial communities; Enables high-throughput and low-cost analysis.Technical problems arise from inherent pitfalls in the databases used for phylogenetic analysis; overestimation of diversity created by incomplete digestion of environmental DNA and the formation of pseudo (TRFs).

Real-time PCR [26]Assesses total microbial communities using probes targeting functional genes; the proportions of specific phylotypes can address metabolic potential of the microbial biomass; has superior sensitivity and is more convenient and less expensive for the quantification of selected bacterial populations; quantification of rRNA directly isolated from ribosomes may be used to reveal the metabolically most active members of a bacterial community.Requires careful calibration; requires extremely accurate controls for inferring cell mass or gene copy; biases introduced by contamination and primer dimers.

MIDI-FAME (Microbial ID, Inc.-fatty acid methyl ester) [27]Creates a complex profile of fatty acids unique to each community sample; useful in comparing one sample with another and in tracking changes in community structure over time or at different sampling locations.Quite limited in the taxonomic information; many fatty acids are common to different microorganisms.

CLPP (community-level physiological profiles of carbon sources) [28, 29]Determine the profile of substrates metabolized by the microbial community; give an estimate of growth and catabolic potential of culturable microorganisms in the original community; relatively inexpensive and commercially are available means of gathering large amounts of information about whole communities of microorganisms.Long procedure; growth dependent, limited in taxonomic information.

Hybridization arrays (microarrays, beadarrays and Phylochips) [30]Detect, identify, and potentially quantify thousands of distinct DNA molecules in a single experiment relatively rapidly and cost-effectively; arrays rely on oligonucleotide probes of 16S rRNA from specific groups of organisms to discern phylogeny, community composition, or function; well suited for identification via multiple-gene functional groups.Limited in exploring entire bacterial diversity. Prior knowledge of the microbial composition is necessary for designing meaningful probes; suffer from cross hybridization between closely related species; genetic variations between strains within species; biases introduced by hairpin structures.

NGS (next-generation sequencing) [31, 32]The longer reads yield more information about the 16S rRNA and thus give a more accurate identification. Allows accurate comparison between environments; large communities can be studied based on phylogeny and/or function; an average sequence depth of 5000 sequences/sample, up to 200 samples, could be sequenced in parallel.Intrinsic sequencing errors; overestimation of taxon abundance; primer pairs greatly influence estimates of microbial community richness and evenness; amplicon products are still subjected to the biases inherent to any PCR-based experiment; the reproducibility of amplicon sequencing across a large number of biological replicates is still questionable.