16S – The oldest, cheapest, and most common method used around the world is known as 16S rRNA. This is a DNA fragment that is unique to bacteria, and the bacteria produce a small subset of the ribosome, the protein-producing factory of the cell. In 1977, a researcher by the name of Carl Woese discovered this fragment and proposed a new division of the bacteria according to the 16S gene sequence. For example, the species of E. Coli has 1500 known varieties, so sequencing by 16S will give us information about the presence of E. Coli but not its varieties.
Pros: Quick and cheap
Cons: Provides information about bacteria only, not about fungi, viruses, and more. In this method, information is obtained only up to the level of the bacterium without knowing which species are in the sample. In addition, the test is prone to bias – bacteria that are high in the microbiome will appear to have an even higher incidence, so it is not certain that their prevalence in the test result represents the true incidence of these bacteria in the intestines.
Shotgun DNA Sequencing – This is an advanced sequencing technology that is widely accepted today, and is the method used by DayTwo. In this method, the DNA of all the microbiome is extracted from the stool sample and broken into small sections by sound waves, from which each part of the strata is taken and eventually 10 million DNA sequences are obtained from one sample of feces. The DNA sequences are compared to well-known gene sequences from bacteria, archaeans, fungi, and viruses from software developed in research labs. This helps identify the details of the entire microbiome at the highest resolution including all of the other microorganisms in the microbiome other than the bacteria. The method allows us to know what they are doing there and what their roles and functions are within the gut.
Pros: Allows the microbiome to be identified in its entirety, up to the highest resolution; also enables us to understand the function of the bacteria.
Cons: This method is more expensive and time-consuming.
Metatranscriptomics – In this method, RNA sequencing of the stool sample is determined. The DNA contains all of the creature’s genes, but not the genes that are currently active. When the gene is active, RNA is produced from it and it then produces protein. The logic behind the RNA test rather than the DNA is that the RNA gives us information about which genes are currently active.
Pros: Provides information about the phenotype – what’s happening in real-time; what the microbiome is currently producing.
Cons: While DNA molecules are stable and DNA can be extracted from ancient animal remains, RNA molecules are very unstable and are prone to rapid degradation. The lifespan of RNA molecules ranges from a few seconds to a few minutes at most. Therefore, in order for the test to be reliable, it must be performed immediately after the stool sample is collected and demands controlled lab conditions. This is unrealistic because when people provide stool samples, it takes time to reach the lab and the RNA can breakdown during this time. Additionally, due to the fact that at any point in time only part of the DNA is transcribed into RNA, the sample will be a snapshot of a momentary situation that does not necessarily reflect the overall activity of the microbiome. Also, most of the RNA produced from the sample is ribosomal RNA so it is difficult to distinguish RNA related to the metabolism of the nutrients.