Metagenomics

Historically, microbial studies have concentrated on pure laboratory cultures. By contrast, metagenomics is defined as the direct analysis of genomes contained in an environmental sample, in order to dillucidate the genomic composition of the entire microbial community ^[1]. Consequently, the descriptions obtained from these kind of studies are more comprehensive than those provided for traditional phylogenetic studies, based on small-subunit ribosomal RNA loci (16S) ^[2].

Besides, shotgun metagenomics provides information about new enzymatic pathways and evolutionary relationships among non-culturable organisms ^[3]. They can be complemented with metatranscriptomic and metaproteomic analyses to determine expression profiles ^[4][5].

In this manner, environments so diverse as skin and guts of animals ^[6][7], marine sediments ^[8], rhizosphere ^[9] and acid mine runoff ^[10] have been studied.

In the last 10 years, metagenomic studies have been impulsed by the development of next generation sequencing (NGS) platforms. Although Sanger sequencing has the lowest error rate and produces longer reads, it has been overcome for NGS technologies, because of its simplicity and low costs ^[11].

The most used NGS platform for metagenomics studies is 454/Roche ^[12][13][10,11]. This system amplifies fragments of DNA adhered to microspheres through a polymerase chain reaction in emulsion. Each microsphere is set in a well and submitted to individual and parallel pyrosequencing, which consist in the sequential addition of the four deoxinucleotide triphosphates. These are incorporated to the coding strand, if they correspond to the sequence, by a DNA polymerase. The subsequent reaction liberates one pyrophosphate, which is consumed in two enzimatic reactions to produce light. Light levels of 1.2 million parallel reactions are detected by a camera and processed to obtain the sequence of the fragments. The reads produced by 454/Roche platform have a mean length of 600 bases, which is ideal for metagenomic studies ^[14].

The enormous amounts of data generated by NGS platforms are subsequently processed with bioinformatic software, in order to assess the taxonomical and functional identity of the DNA sequenced. This step is called binning and it is generally done by sequence similarity ^[15]. Although there are several tools to perform a metagenomic analysis, the most widely used are the MG-RAST server ^[16] and metAMOS ^[17].