16S sequencing vs. Shotgun metagenomics: Which one to use when it comes to microbiome studie

While a lot of attention has been paid in recent years to the advances made in sequencing the human genome, next-generation sequencing has also led to an explosion of sequencing used to study microbiomes. There are two common methods of sequencing performed to study the microbiome: 16S rDNA sequencing and shotgun metagenomics.

What is 16S sequencing?

The 16S ribosomal gene is thought to exist in all bacteria, but still has regions that are highly variable between species. Because of this, primers have been created to amplify conserved regions that surround variable regions, allowing researchers to target the areas of the genes that are similar to observe the areas that are distinct. Because this approach allows us to observe very specific regions of the genome, we can drop the sequencing needed per sample dramatically, only needing around 50,000 – 100,000 reads to identify different bacterial species in a sample.

The main drawback of this technique is that it can only identify bacteria, and does not identify viruses or fungi.

What is shotgun metagenomics?

Shotgun metagenomics surveys the entire genomes of all the organisms present in the sample, as opposed to only the 16S sequences. One of the main advantages of this over 16S sequencing is that it can capture sequences from all the organisms, including viruses and fungi, which cannot be captured with 16S sequencing. Additionally, it’s less susceptible to the biases that are inherent in targeted gene amplification.

Perhaps most interestingly, it can also provide direct information about the presence or absence of specific functional pathways in sample, also known as the ‘hologenome’. This can provide potentially important information about the capabilities of the organisms in the community. Furthermore, shotgun metagenomics can be used to identify rare or novel organisms in the community, which 16S cannot do.

So which one should I use?

Like anything else, it really depends. 16S studies can be incredibly useful for comparison across different samples (like different environments, or different time points). And some studies have found that 16S sequencing is superior in these types of studies for identifying a higher number of phyla in a particular sample [1], while other studies have of course found the exact opposite [2].

When it comes down to it, it’s really important to evaluate your project needs carefully depending on what you’re trying to accomplish. For example, a large scale project that looks to examine hundreds of samples in order to evaluate the differences in microbiota across different environments might very well prefer to use 16S sequencing, since it is so much more cost-efficient than metagenomics sequencing. On the other hand, a project that is looking to deeply investigate a smaller number of samples might be a better candidate for metagenomics sequencing, which would allow them uncover all the organisms that are present in a particular sample (including viruses and fungi), as well as identify the most dominant gene pathways that are present in that particular sample.

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