To date, Next Generation Sequencing (NGS) techniques remain the most commonly used methodologies for studying the microbiome, a major subject of scientific research in recent years. Among the thousands of articles published each year (illustration 1), two main categories of analysis emerge: 16S metabarcoding (also called 16S metasequencing or targeted metagenomics) and shotgun metagenomics. Both approaches are based on the analysis of microbiome DNA extracted from the biological sample, but each presents distinct advantages and disadvantages. This article offers a detailed comparison of these two techniques to better understand their applications, advantages, and limitations.
16S Metabarcoding
Starting from the total DNA extracted from a biological sample, 16S metabarcoding focuses on the amplification and sequencing of a specific region of the gene coding for 16S ribosomal RNA, which is highly conserved in bacteria and archaea (illustration 2). This region contains variable segments that allow for the identification and classification of microorganisms, and depending on the PCR primers used, all or part of this approximately 1500 base pair gene is sequenced. A metabarcode analysis produces a microbiota profile. In practice, this profile is presented in the form of a table of relative abundance of the different bacteria identified.
Advantages of 16S Metabarcoding
1. Quantity/Quality of DNA Required: Due to the PCR amplification step, metabarcoding requires only a small amount of DNA (less than 1 ng can yield a fully exploitable profile) and is not very sensitive to the presence of host DNA (which will not be amplified).
2. Taxonomic Specificity: The 16S rRNA gene is a well-established phylogenetic marker, allowing precise identification of bacteria down to the genus and sometimes species level.
3. Cost and Simplicity: 16S metabarcoding is generally less expensive and less complex to implement than shotgun metagenomics. Amplification and sequencing protocols are well-documented and often standardized.
4. Diversity Analysis: This method allows for a rapid analysis of microbial diversity within a sample, particularly through various alpha and beta diversity indices, which is useful for obtaining an overview of the bacterial community under study.
Limitations of 16S Metabarcoding
1. Analysis Bias: PCR amplification can introduce biases, favoring certain groups of bacteria over others, leading to false positives, false negatives, or erroneous profiles. These biases are due to the presence of multiple copies of 16S in some bacteria, the amplification of contaminating DNA from the environment or reagents, and incorrect sequence assignments.
2. Limited Resolution: Although effective for taxonomic identification to the genus level, the method has limitations for distinguishing closely related species and does not allow for the study of genomic functions unless using bioinformatic derivations (e.g. https://picrust.github.io/picrust/).
3. Exclusion of Non-Bacteria: 16S metabarcoding targets only bacteria and archaea, neglecting viruses, fungi, and other microorganisms present in the microbiome. However, interactions between different kingdoms are important, for example, concerning the vaginal flora where relationship between bacteria and yeast is a key component of health status.
Shotgun Metagenomics: Presentation and Applications
Starting from the same total DNA extracted from a biological sample, shotgun metagenomics involves fragmenting the entire DNA and randomly sequencing the resulting fragments (illustration2). Unlike 16S metabarcoding, this method does not use PCR and thus allows for a more comprehensive analysis of the microbial composition.
Advantages of Shotgun Metagenomics
1. Comprehensive Analysis: Shotgun metagenomics allows for the identification of all types of DNA-containing microorganisms in a sample, including bacteria, archaea, viruses, fungi, protozoa, etc.
2. Fine Taxonomic Resolution: This method offers a finer taxonomic resolution, allowing identification down to the species, or even strain level.
3. Genomic Function Study: Sequencing from any part of the microbial genome allows for the identification of functional genes, providing information on the metabolic capabilities of microorganisms.
4. Avoiding PCR Bias: Since shotgun metagenomics does not use PCR, it avoids the amplification biases associated with 16S metabarcoding, particularly the impact of contamination from the environment or laboratory reagents.
Limitations of Shotgun Metagenomics
4. Quantity/Quality of DNA Required: Without targeted amplification of microbiota DNA, shotgun metagenomics requires a larger amount of DNA (greater than 1 ng) and is sensitive to the presence of host DNA.
2. Cost and Complexity: Shotgun metagenomics is a little more expensive and above all technically more complex. It requires high-throughput sequencing capabilities and sophisticated bioinformatic analyses that demand significant computational power and storage capacity.
3. Data Overload: Shotgun metagenomics produces a lot of data from a single sample. Taxonomic tables for bacteria, archaea, eukaryotes, viruses, and functional tables according to different reference systems (https://geneontology.org/, https://enzyme.expasy.org/) require the use of more complex and innovative statistical analysis methods to derive significant elements, particularly when microbiome data are combined with other clinical data and because of the compositional nature of the data.
Use Cases and Practical Applications
16S Metabarcoding
– Ideal for exploratory studies of bacterial composition and diversity.
– Recommended for matrices with very low microorganism density (compensated by the PCR amplification step), such as certain areas of the skin.
Shotgun Metagenomics
– Ideal for exploring microbiomes in their entirety (bacteria and yeast/fungi and viruses…).
– Recommended for projects requiring species-level resolution and exploration of functional aspects.
Conclusion
16S metabarcoding is an effective method for rapid and economical analyses of bacterial diversity, while shotgun metagenomics offers a more comprehensive and detailed overview of microbial communities, including their genomic functions.
The choice between 16S metabarcoding and shotgun metagenomics ultimately depends on the specific endpoints/objectives of the study, the available resources, and the required level of detail. This choice also involves considering sequencing depth and the overall study design, including inclusion and exclusion criteria, the duration of different phases, sampling points, and more. All this requiring the support of a specialist in microbiome analysis.
Illustration 1
Illustration 2