My own personal opinion is that we will really only begin to do meaningful microbial ecology when sequencing methods are invented that provide us with very long sequences.
More recently, with the introduction and growth of molecular tools in microbial ecology, many culture-independent methods have developed to overcome the cultivation biases and allow detailed information on microbial community diversity, structure, and function.
The analysis using 16S rRNA genes as a phylogenetic marker was a revolutionary strategy for microbial ecology with cultured-independent method being developed since 90s, after the work of Lane and collaborators [ 44].
Special emphasis is placed here on the need for precise, rather than unbiased, estimation methods in microbial ecology, but precision can be judged only with a very large sample or with multiple samples drawn from a single community.
The recent introduction of next generation sequencing methods in microbial ecology has offered a powerful alternative to traditional clone library-based and microarray assessments of microbial diversity.
I hope this manuscript encourages more discussion of microbial ecology and in particular the methods and what the experiments really mean.
Established methods of microbial ecology that allow the direct measurement of metabolic conversions in natural microbial samples from humans under different experimental conditions, such as incubation with isotopically-labelled substrates, dye probes for specific compounds combined with microscopy or electrochemical microsensors, are rarely reported.
NMDS does not need the assumption of linear associations among variables being described as the most efficient ordination method for microbial ecology [ 29].
Using the 16S ribosomal RNA gene as a phylogenetic marker was a real breakthrough for microbial ecology studies, with several culture-independent methods being developed since Pace et al. [ 72] proposed the direct cloning of environmental DNA.
Our results demonstrate the applicability of barcoded pyrosequencing as a high-throughput method for comparative microbial ecology.
