Are you planning a microbiome study? If so, you might have thought about whether you should use 16S rRNA sequencing or shotgun sequencing. We want to show the differences between the two methods, as well as their strengths and weaknesses.
Microorganisms are the most abundant cellular life form, occupying numerous metabolic niches – from the deep sea to the remotest desert. 16S rRNA sequencing is a common way to study metagenomic communities. However, metagenomic shotgun sequencing is gaining increasing attention. Consider the strengths and weaknesses of every approach to decide which method is best for your project.
First, let’s look at the 16S rRNA sequencing method. It targets the hypervariable regions (V1-V9) of the 16S rRNA gene. Most 16S sequencing approaches do not cover all regions. In contrast, full-length 16S sequencing allows the analysis of the whole 16S gene with all hypervariable regions. The amplification is performed using PCR, followed by library preparation and sequencing. After sequencing, the data can be evaluated using well-established reference databases. The method gives insights into the sample’s composition. Organisms can be identified down to genus or even species level. However, the 16S gene can only be found in bacteria and archaea, which means the method is limited to these microbial domains. Full-length 16S sequencing is further limited to bacteria due to the PCR primers.
Compared to this targeted approach, shotgun metagenomic sequencing gets its name as it randomly breaks the entire DNA of a microbial sample into many tiny fragments. The fragmentation is done via mechanical shearing. After the fragmentation step, sequencing libraries are prepared. Databases are available to evaluate the sequencing results. These databases are, unfortunately, not complete for most ecosystems. To identify unknown, non-cultivable microorganisms, de-novo assembly can be performed. Analyzing the whole genome of the microbes further allows us to gain insights into the individual microbial functional properties. Shotgun metagenomic sequencing is not limited to one microbial domain in the sample but allows the simultaneous detection of bacteria, fungi, viruses, and various other microorganisms. Figure 1 illustrates the different approaches of the two described methods.
Let us now consider the methods’ strengths and weaknesses. We will focus on the different possible sample types, the composition of the sample and its function, the possible analyses, and the data capacity: More complex sample types benefit from shotgun sequencing. For less complex samples with known bacterial species and a general interest in the exact composition and proportions of the species, 16S sequencing is a good choice. Furthermore, one must ask oneself how exactly the composition shall be determined. Is a statement at the genus level sufficient, does it need to go down to the species level, or is it even a strain-specific approach? Should the function of the individual organisms be examined in addition to the composition? The informative value of shotgun metagenomic sequencing is higher than that of 16S sequencing. Additionally, it must be considered that processing and interpreting shotgun metagenomics data entails more data capacity and experience.
With this information, you are ready for your next metagenomic project. CeGaT offers both Full-Length 16S Sequencing and Shotgun Metagenomic Sequencing. Don’t hesitate to unravel your sample’s diversity!
Figure 1 | Comparison of the 16S sequencing approach and the shotgun metagenomic sequencing approach. In 16S sequencing, only bacterial species can be analyzed. For shotgun metagenomic sequencing, a multitude of different microorganisms can be analyzed.