Bioinformatic methods for functional metagenomics.

<p>Studies that aim to define the composition and function of uncultured microbial communities are often referred to collectively as “metagenomic,” although this refers more specifically to particular sequencing-based assays. First, community DNA is extracted from a sample, typically uncultured, containing multiple microbial members. The bacterial taxa present in the community are most frequently defined by amplifying the 16S rRNA gene and sequencing it. Highly similar sequences are grouped into Operational Taxonomic Units (OTUs), which can be compared to 16S databases such as Silva <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002808#pcbi.1002808-Pruesse1" target="_blank">[16]</a>, Green Genes <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002808#pcbi.1002808-DeSantis1" target="_blank">[14]</a>, and RDP <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002808#pcbi.1002808-Cole1" target="_blank">[15]</a> to identify them as precisely as possible. The community can be described in terms of which OTUs are present, their relative abundance, and/or their phylogenetic relationships. An alternate method of identifying community taxa is to directly metagenomically sequence community DNA and compare it to reference genomes or gene catalogs. This is more expensive but provides improved taxonomic resolution and allows observation of single nucleotide polymorphisms (SNPs) and other variant sequences. The functional capabilities of the community can also be determined by comparing the sequences to functional databases (e.g. KEGG <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002808#pcbi.1002808-Kanehisa2" target="_blank">[170]</a> or SEED <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002808#pcbi.1002808-Overbeek1" target="_blank">[171]</a>). This allows the community to be described as relative abundances of its genes and pathways. Figure adapted from <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002808#pcbi.1002808-Morgan1" target="_blank">[172]</a>.