In this article, we’ll take a closer look at the two most commonly used methods for analysing the microbiome, 16s rRNA testing and shotgun metagenomic sequencing. We’ll explain what microbiome sequencing is and why it matters, as well as how each method works and what kind of information they can provide. You’ll also find a clear comparison of the key differences between the two. Whether you're exploring microbiome testing for research, clinical use or personal health, this guide will help you understand which method may be more suitable for your needs.

Here are the five biggest takeaways from this article about Shotgun Metagenomic Sequencing vs 16s rRNA testing:

• 16S rRNA testing focuses only on bacteria and gives a broad overview, while shotgun metagenomic sequencing looks at all types of microbes, providing much more detailed results.

• Shotgun metagenomic sequencing can identify microbes down to species or strain level and give insight into what those microbes might be doing, based on their genes.

• 16s rRNA is easier to interpret, but it can miss important details because it only targets a single gene found in bacteria.

• Shotgun sequencing is more advanced, while 16S rRNA is more beginner-friendly and uses well-established databases.

• If you're looking for a quick and general view of the bacteria in your gut, 16S rRNA may be enough. But if you want a deeper, more complete understanding of your entire microbiome, shotgun metagenomics is likely to be the better option.

Understanding the microbiome has become much more accessible in recent years, thanks to major advances in DNA sequencing technology. In the year 2000, sequencing the entire human genome cost around $100 million. By 2020, that cost had dropped to roughly $1,000. This reduction in cost and increase in speed has opened the door to progress in microbiome research across human, animal and environmental studies. 

Two of the most widely used approaches are 16S rRNA testing and shotgun metagenomic sequencing. Understanding how these techniques are different, and the benefits and limitations of each, can help you understand which provider of testing is best for you and your needs.

In the video below by Microbiome Insights, Dr. Ruairi Robertson explains the pros and cons of each form of gut testing.

Overview

16S rRNA testing is a widely used method for examining the gut microbiome, with a specific focus on bacteria. It works by targeting the 16S ribosomal RNA gene, a part of bacterial DNA that is present in all bacteria but not in other microbes such as viruses or fungi. This makes it useful for identifying the different types of bacteria living in the gut and understanding their general abundance. While it gives a broad overview of the bacterial community, it does not provide information about other types of microbes, and its ability to identify bacteria at a detailed level is limited.

The process for 16S rRNA Sequencing gut testing

The 16S rRNA sequencing process begins by collecting a sample, typically a stool sample in gut microbiome studies. The bacterial DNA is then extracted from the sample and the 16S rRNA gene is amplified using a method called polymerase chain reaction (PCR). This amplified gene is then sequenced and compared to reference databases to match it with known bacterial groups. The result is a profile showing which types of bacteria are present and in what general quantities. However, this method usually cannot identify bacteria at the species or strain level, which means the results are less detailed than other sequencing methods.

The benefits

One of the main benefits of 16S rRNA testing is that it offers a relatively quick way to study the bacterial component of the microbiome. It’s especially useful for identifying dominant bacterial groups and spotting general imbalances in the gut. Because it focuses solely on bacteria, the analysis is simpler and less data-heavy compared to more comprehensive methods. 

As a result, it remains a popular choice for many commercial and academic microbiome studies. That said, it is increasingly being viewed as outdated when compared to more advanced techniques like shotgun metagenomic sequencing, which provide a fuller picture of the entire microbiome, not just the bacteria.

Overview

Shotgun Metagenomic Sequencing is a highly advanced technique used to study the gut microbiome in great detail. Unlike more targeted methods, this approach examines all the genetic material present in a stool sample. That includes not only bacteria, but also viruses, fungi and other types of microbes. Because it looks at the full range of DNA, it provides a much broader and more detailed view of the gut microbiome and how it’s functioning.

The process for Shotgun Metagenomic Sequencing gut testing

To carry out this form of testing, a small stool sample is collected and processed in a laboratory. The DNA is extracted from the sample and then broken into smaller fragments. These fragments are then sequenced, meaning the exact order of the DNA bases (A, T, C and G) is read. Once this raw data has been collected, it is compared to reference databases to identify which microorganisms are present, down to the species or even strain level. This process gives a detailed and accurate picture of your gut microbiome.

The benefits

One of the main advantages of Shotgun Metagenomic Sequencing is its level of detail. It doesn’t just show what types of bacteria are present, it also identifies other important microbes such as fungi and viruses. This means it can offer a more complete view of your gut health. It is also highly accurate and capable of detecting organisms at the species or strain level, which is particularly useful for understanding how your microbiome might be affecting your overall wellbeing. This level of detail can help provide more personalised insights, making it a valuable option for anyone seeking a deeper understanding of their gut health. It is the method used by Vivere for this very reason.

When looking at the differences between 16S rRNA testing and shotgun metagenomic sequencing, it helps to know what each test can actually show. Both use a stool sample and involve a similar level of preparation, but what they reveal about your microbiome is quite different. 

16S rRNA testing focuses only on bacteria and a group called archaea and usually only identifies them at a general (genus) level. Sometimes it can go further and identify the exact species, but this depends on which part of the DNA is tested. Shotgun metagenomic testing, on the other hand, looks at all the DNA in the sample. This means it can spot not just bacteria, but also viruses, fungi and other microbes, often down to the exact species or even strain, if enough DNA is read.

Another important difference is what the results can tell you. Shotgun testing gives you an idea of what the microbes might be doing in your gut, based on the genes they carry. This is known as functional profiling. 16S rRNA testing can't really do this, although it can make rough guesses. Shotgun testing does require more advanced computer analysis and uses newer reference databases that are still growing. It’s also more likely to pick up bits of your own DNA by mistake, though this can be managed. 

16S rRNA testing is easier to analyse but can be less accurate, as it’s more affected by certain steps in the process. 

Overall, shotgun metagenomic testing gives a more detailed and complete picture of the gut microbiome, but it is more complex and requires more resources to carry out. The table below outlines the main differences between the two testing methods, as we have just explained.

Here are the five biggest differences between 16S rRNA testing and shotgun metagenomic sequencing:

What they detect

• 16S rRNA testing only identifies bacteria and archaea.

• Shotgun metagenomics can detect all types of microbes, including bacteria, viruses, fungi and parasites.

Level of detail

• 16S rRNA usually identifies microbes at the genus level and only sometimes at the species level.

• Shotgun metagenomics can go down to the species or even strain level, offering much more precise results.

Functional insights

• 16S rRNA doesn’t directly show what the microbes are doing in the body, only rough predictions are possible.

• Shotgun metagenomics provides information on the functions and roles of microbes, based on the genes they carry.

Types of data and analysis needed

• 16S rRNA is simpler to analyse and suitable for those with basic to intermediate scientific knowledge.

• Shotgun metagenomics needs more advanced analysis tools and expertise, due to the large and complex data it generates.

Bias and accuracy

• 16S rRNA is more prone to bias, as it relies on amplifying a specific gene, which can affect which microbes are detected.

• Shotgun metagenomics has lower bias and tends to provide a more accurate and complete picture of the microbiome.

Both 16S rRNA testing and shotgun metagenomic sequencing offer insight into the gut microbiome, but they differ significantly in what they can detect and the level of detail they provide. 16S rRNA testing is simpler and effective for identifying general patterns in bacterial populations. However, it is limited in scope and detail. 

Shotgun metagenomic sequencing, while more complex, delivers a far more comprehensive view by detecting all types of microbes, including viruses and fungi, and offering deeper insights into their potential functions.

Vivere helps you take control of your health with personalised insights from state-of-the-art gut microbiome testing, nutritional guidance, science-backed supplements and expert support. Sign up today and start living better, for longer.

Validation and Retrospective Clinical Evaluation of a Quantitative 16S rRNA Gene Metagenomic Sequencing Assay for Bacterial Pathogen Detection in Body Fluids - ScienceDirect