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Metagenomics is the study of microbial communities in their original habitats and gives a comprehensive insight into the biochemical and metabolic interactions within these communities. Metagenomics can also help identify individual species within microbial habitats with no pre-isolation required. Metagenomics methods are often employed to compare differentially expressed genes within various functional pathways across alternative environments. It reveals the adaptive mechanisms of microorganisms under different environmental stresses and explores the interactions between them and other components of their surroundings. There are two main approaches used in metagenomic studies – shotgun-based and amplicon-based. But what exactly are these and how do you decide which to use for your research goals?
16S/18S/ITS Amplicon-based metagenomic sequencing
Amplicon-based metagenomic sequencing efficiently screens for variants and target organisms to describe and compare the diversity of multiple complex environments. The approach is frequently used in population and community microbial ecology studies, phylogenetic reconstruction of target microbial groups, identification of individual species in mixed cultures, and detection of organisms of interest, both pathogenic and beneficial.
Amplicon-based metagenomics exploits conserved regions within ribosomal RNA known as amplicons that provide a template for the design of primers to study the variable regions between them. These variable regions are specific to a genus and sometimes a species, meaning that with this method microorganisms can be reliably identified at the genus levels, and some at species levels. The conserved regions used are 16S rRNA, widely used to identify bacteria and archaea; 18S rRNA to identify microbial eukaryotes such as fungi and protists; and ITS sequencing, the preferred method of identification for fungal species.
Shotgun-based metagenomic sequencing
Shotgun-based metagenomic sequencing provides information on the total genomic DNA from all organisms in a sample, avoiding the need for isolation and cultivation of microorganisms or amplification of target regions. This is crucial because it is believed that nearly 99% of all microorganisms cannot be cultivated in the laboratory. Shotgun metagenomic sequencing uses next-generation sequencing to provide information on the genetic diversity of host-associated microbial communities, the functional diversity of microbial communities, gene prediction and annotation, host-microbe interactions, and microbiota-based disease mechanisms.
Metagenomic shotgun sequencing involves randomly shearing the DNA of the microbial genome into small fragments, then adding a universal primer at both ends of the fragments for PCR amplification and sequencing. The sequence of the small fragments is then spliced into a longer sequence through assembly. Because this method sequences the full genome it can provide information to determine not only genus and species but also subspecies and strains in some cases. It can also analyse gene expression and function and how these metabolic functions contribute to community fitness and host-microbe interactions and symbiosis.
Which to use
Which method to use depends on your research goals. Do you want to identify what’s there, or do you want to learn about the functions of what’s there? For example, a large-scale project with the aim of identifying the composition of communities across a range of environments or conditions would likely benefit from amplicon-based sequencing, since it is a much more cost-efficient method than shotgun-based metagenomics and won’t provide metabolic function analysis superfluous to requirement. Amplicon-based sequencing strategies are designed mainly for the purpose of studying the phylogenetic relationship of species, the species composition, and the biodiversity of a microbial community. Besides cost efficiency, other advantages of amplicon-based sequencing include resistance to host DNA contamination, and the risk of false positives is relatively low. The flipside of this is that the resolution is lower and functional profiling is not available.
On the other hand, if your goal is to identify the microorganisms present along with analysing the metabolic functions and dominant pathways within the community, shotgun-based metagenomics would be the approach for you. Apart from the taxonomic analysis that amplicon-based sequencing can provide, shotgun sequencing can also conduct in-depth research on genes and functions of a microbial community, such as pathway analysis using KEGG and GO. This functional profiling comes alongside other advantages such as high resolution, novel gene detection and the estimation of the presence and absence of certain genes and functions. Because of this enriched analysis, shotgun-based sequencing is the more expensive option of the two methods and is also more susceptible to interference from host DNA contamination. It is recommended that host DNA is removed to avoid any extra sequencing costs.
Overall, if your goal is to sequence a large number of microbiome samples across different environments or conditions and analyse the diversity of the communities, amplicon-based sequencing is the approach for you. If you need to analyse the metabolic and biochemical functions within your microbiome samples, shotgun-based sequencing is required.
Eager to learn more? Check out our previous post for more insights!
What you can explore with non-coding RNA data
WGS vs WES: Which Genetic Sequencing Method is Right for You?
Expanding Horizons in Genomic Research with Long-Read Sequencing
A Basic Guide to RNA-sequencing
Uncovering the Genetic Basis of Rare and Complex Diseases through Whole Genome Sequencing (WGS)
In the Lab: A Closer Look at DNA Methylation Sequencing Techniques
Long-read Sequencing Technology Explained
How to Choose Normalization Methods (TPM/RPKM/FPKM) for mRNA Expression
WGBS vs RRBS
16S/18S/ITS Amplicon-based metagenomic sequencing
Amplicon-based metagenomic sequencing efficiently screens for variants and target organisms to describe and compare the diversity of multiple complex environments. The approach is frequently used in population and community microbial ecology studies, phylogenetic reconstruction of target microbial groups, identification of individual species in mixed cultures, and detection of organisms of interest, both pathogenic and beneficial.
Amplicon-based metagenomics exploits conserved regions within ribosomal RNA known as amplicons that provide a template for the design of primers to study the variable regions between them. These variable regions are specific to a genus and sometimes a species, meaning that with this method microorganisms can be reliably identified at the genus levels, and some at species levels. The conserved regions used are 16S rRNA, widely used to identify bacteria and archaea; 18S rRNA to identify microbial eukaryotes such as fungi and protists; and ITS sequencing, the preferred method of identification for fungal species.
Shotgun-based metagenomic sequencing
Shotgun-based metagenomic sequencing provides information on the total genomic DNA from all organisms in a sample, avoiding the need for isolation and cultivation of microorganisms or amplification of target regions. This is crucial because it is believed that nearly 99% of all microorganisms cannot be cultivated in the laboratory. Shotgun metagenomic sequencing uses next-generation sequencing to provide information on the genetic diversity of host-associated microbial communities, the functional diversity of microbial communities, gene prediction and annotation, host-microbe interactions, and microbiota-based disease mechanisms.
Metagenomic shotgun sequencing involves randomly shearing the DNA of the microbial genome into small fragments, then adding a universal primer at both ends of the fragments for PCR amplification and sequencing. The sequence of the small fragments is then spliced into a longer sequence through assembly. Because this method sequences the full genome it can provide information to determine not only genus and species but also subspecies and strains in some cases. It can also analyse gene expression and function and how these metabolic functions contribute to community fitness and host-microbe interactions and symbiosis.
Which to use
Which method to use depends on your research goals. Do you want to identify what’s there, or do you want to learn about the functions of what’s there? For example, a large-scale project with the aim of identifying the composition of communities across a range of environments or conditions would likely benefit from amplicon-based sequencing, since it is a much more cost-efficient method than shotgun-based metagenomics and won’t provide metabolic function analysis superfluous to requirement. Amplicon-based sequencing strategies are designed mainly for the purpose of studying the phylogenetic relationship of species, the species composition, and the biodiversity of a microbial community. Besides cost efficiency, other advantages of amplicon-based sequencing include resistance to host DNA contamination, and the risk of false positives is relatively low. The flipside of this is that the resolution is lower and functional profiling is not available.
On the other hand, if your goal is to identify the microorganisms present along with analysing the metabolic functions and dominant pathways within the community, shotgun-based metagenomics would be the approach for you. Apart from the taxonomic analysis that amplicon-based sequencing can provide, shotgun sequencing can also conduct in-depth research on genes and functions of a microbial community, such as pathway analysis using KEGG and GO. This functional profiling comes alongside other advantages such as high resolution, novel gene detection and the estimation of the presence and absence of certain genes and functions. Because of this enriched analysis, shotgun-based sequencing is the more expensive option of the two methods and is also more susceptible to interference from host DNA contamination. It is recommended that host DNA is removed to avoid any extra sequencing costs.
Overall, if your goal is to sequence a large number of microbiome samples across different environments or conditions and analyse the diversity of the communities, amplicon-based sequencing is the approach for you. If you need to analyse the metabolic and biochemical functions within your microbiome samples, shotgun-based sequencing is required.
Eager to learn more? Check out our previous post for more insights!
What you can explore with non-coding RNA data
WGS vs WES: Which Genetic Sequencing Method is Right for You?
Expanding Horizons in Genomic Research with Long-Read Sequencing
A Basic Guide to RNA-sequencing
Uncovering the Genetic Basis of Rare and Complex Diseases through Whole Genome Sequencing (WGS)
In the Lab: A Closer Look at DNA Methylation Sequencing Techniques
Long-read Sequencing Technology Explained
How to Choose Normalization Methods (TPM/RPKM/FPKM) for mRNA Expression
WGBS vs RRBS