Introduction to Sequencing

You may be wondering how we are able to detect all the different kinds of bacteria we receive in your sample. Differentiating between all the different types of organisms in the gut sounds like an impossible task. However, once you understand the basics of how this technology works, everything will seem a lot more simple.

What is rRNA?

The foundation of Mycrobiome’s sequencing approach utilizes a molecule known as ‘16S rRNA’. The RNA most people know about is essentially a messenger molecule in the cell. This molecule is a copy of genetic instructions that codes for an essential protein. However, rRNA does not work in this way. This molecule is special because it exists as part of the ribosome (the protein assembling machine). The rRNA is responsible for binding the ribosome to the correct amino acids in the correct order. The ‘16S’ part of the name simply refers to the length of the molecule in humans, rRNA is typically found in 18S and 28S lengths. Only bacteria have 16S sized rRNA.

Why use rRNA?

Because rRNA is something all bacteria need, there is a lot of similarity between the rRNA in different species of bacteria. In fact, there are certain regions of the rRNA that do not change at all because they are so essential that any changes in the RNA sequence would kill the bacteria. However, between these essential sections, there are regions known as variable regions. These areas of the rRNA molecule do not need to be perfectly conserved for proper functioning of the ribosome. As a consequence, mutations slowly change the sequences in these areas. Now here is the cool part; by comparing the changes in the sequences in variable regions, you can see how closely related different bacteria are.

For example, if you look at two bacteria and they have variable regions that are 90% similar, they are more closely related than bacteria that have 80% overlap in their variable regions. This makes logical sense; if two bacteria are distantly related, lots of time has passed since they split from each other. Thus, there has been a lot of time for random mutations to build up in the variable regions of their rRNA molecules, reducing the overlap percentage. Scientists have compared lots of bacteria like this and assigned certain mutation patterns to certain phyla (bacteroidetes, firmicutes, etc.).

Advantages of 16S rRNA Sequencing

1. The main advantage of using 16S rRNA sequencing is that all bacteria have this molecule. This means that using this technique will capture the entire population of bacteria in a sample, providing a more accurate analysis.
2. Additionally, all bacteria have many copies of the 16S rRNA gene in their genomes. This means that it is easier to isolate these target genes for sequencing (we sequence the DNA gene from which the rRNA is created) without getting too much interference from random sequences.
3. Unlike genes in plasmids, bacteria do not swap rRNA genes very often, which makes it easy to pinpoint what phylum/genera each bacteria belongs in without much room for misidentification.
4. Finally 16S rRNA is one of the cheaper methods by which to sequence the microbiome, keeping costs as low as possible.

Disadvantages of 16S rRNA Sequencing

1. While it is true that all bacteria have 16S rRNA, not all bacteria have the same number of copies of rRNA genes. This means it is difficult to determine exactly how many of each bacteria are present in your gut. For example, a single bacteria with 100 copies of a rRNA gene will show up as 100 times more abundant than a bacteria with a single copy (I made up these numbers). On average, it is possible to determine general trends in relative population but the margins of error are high.
2. Another issue of 16S rRNA sequencing is the specificity of its phylogenetic classifications. This method is able to separate bacteria into phyla and occasionally genera, but it cannot distinguish between species. This is because changes in species may occur due to mutations in different parts of the genome that are not getting sequenced. Two different species of bacteria that have not developed any mutations in their 16S rRNA genes will therefore show up as the same species.

Conclusion

All in all, 16S rRNA sequencing is a good candidate for large scale microbiome analyses. It enables us to take lots of customer samples and provide reliable data quickly and economically. The technology itself uses simple concepts of genetic amplification and sequencing to identify all the different microbes in your samples, essentailly tracking the age old process of evolution thorugh genetics. Hopefully, this blog post helped you better understand the basics of how this technology works.