HOW IS THE MICROBIOME STUDIED?
The microbiome of any given tissue comprises hundreds to thousands of species, the majority of which cannot be cultured, that is, grown in isolation in a Petri dish. To decipher the various species, samples are usually collected using a sterile cotton swab or spatula and the total collection of DNA from organisms attached to the swab/spatula is isolated. The general types of bacterial species can be elucidated by analyzing a specialized part of the genome that is involved in making a critical component of the ribosome, which is a key part of our protein synthesis machinery. This critical component is the 16S ribosomal RNA gene (rDNA). The 16S rDNA gene is highly conserved but has several differences that are unique to each of the many types of bacteria. The relevant parts of the 16S rRNA genes are amplified and sequenced and the types and numbers of bacteria present in each complex mixture are deduced from the characteristic 16S rDNA of that group of bacteria. The abundance of each bacterial family is derived from the number of times that particular type of 16S rDNA is sequenced.
This type of analysis usually identifies the general families of bacteria and can sometimes reveal the individual species. Although very powerful in answering the question of, “What general types of bacteria there?”, this analysis does not answer the questions of, “What exact bacteria are there?” and “What are they doing?” In order to identify individual species and many of the biochemical pathways that are present, many more millions of DNA fragments are sequenced using the latest high throughput DNA sequencing technologies—in fact, the very same ones used to sequence human genomes. These technologies generate a collection of a few hundred base snippets of information across the entire genome of the different bacteria as well as any other organisms in the sample, including viruses, fungi, and other single- celled organisms. The snippets can be assembled into larger segments, and the exact species, their frequencies, and the biochemical pathways they encode are elucidated. The composition of complex samples containing hundreds to thousands of different species can then be deciphered.