Overview of methods for community profiling and functional metagenomics. Patient tissue samples contain a mixture of human and microbial DNA. Microbial DNA is derived from a community of bacteria and other organisms present at their relative abundance in the sample, indicated here using different colors. Once DNA has been extracted from the sample, two metagenomic methods can be applied. In functional metagenomics the total DNA is sequenced and analyzed by comparing it to databases of known genomes (for example, NCBI and IMG) and 16S rRNA genes (for example, RDP, Green Genes and Silva) to identify bacterial taxa and their abundance. Sequences are also compared to known proteins (for example, SIMAP, MG-RAST, KEGG) for functional analysis of genes, pathways and relative frequency. In community profiling, hypervariable regions of the 16S rRNA gene from bacteria are amplified and sequenced. Highly similar sequences are binned by operational taxonomic units and compared to databases of 16S rRNA genes from known bacteria (for example, RDP, Green Genes and Silva) to identify bacterial taxa and their frequency. 16S rRNA gene sequences can be used in subsequent analyses of phylogenetic diversity in the sample. IMG: Integrated Microbial Genomes; KEGG: Kyoto Encyclopedia of Genes and Genomes; MG-RAST: Metagenomic Rapid Annotations using Subsystems Technology; NCBI: National Center for Biotechnology Information; OTU: Operational Taxonomic Unit; RDP: Ribosomal Database Project; SIMAP: Similarity Matrix of Proteins.