Amplicon vs shotgun sequencing

Metagenomics delves into the study of microbial communities directly within their natural environments, offering a thorough understanding of the biochemical and metabolic interactions that occur within these ecosystems. This approach also allows for the identification of individual species within microbial habitats without the need for prior isolation. By utilising metagenomics, researchers can uncover the types of microorganisms present, along with their functions and interactions. Metagenomic studies typically utilise two main approaches: shotgun-based and amplicon-based. But what do these methods entail, and how should you choose the one that aligns with your research objectives?

16S/18S/ITS Amplicon-Based Sequencing

Amplicon-based sequencing is a popular method for studying microbial diversity and community composition. This technique is widely applied in studies of microbial ecology, population dynamics, phylogenetic analysis of specific microbial groups, and the identification of species in mixed cultures, including pathogenic or beneficial organisms.

Amplicon-based sequencing targets conserved regions within ribosomal RNA genes to design primers that amplify the variable regions.. These variable regions are unique to certain genera and sometimes species, enabling reliable identification at the genus level, and occasionally at the species level. The regions targeted for various domain types are as follows:

• 16S rRNA: Used for identifying bacteria and archaea.
• 18S rRNA: Applied to microbial eukaryotes, including fungi and protists.
• ITS Sequencing: Preferred for identifying fungal species.

This method excels in profiling microbial diversity and phylogeny across different environments and is often used to study species in mixed cultures

Shotgun-Based Metagenomic Sequencing

Shotgun-based metagenomic sequencing offers a comprehensive analysis of the total genomic DNA from all organisms in a sample, eliminating the need for microbial isolation, cultivation, or amplification of specific regions. This is particularly important as nearly 99% of microorganisms cannot be cultivated in laboratory settings. By utilising next-generation sequencing, shotgun metagenomics provides insights into the genetic diversity of microbial communities associated with hosts, their functional diversity, gene prediction and annotation, host-microbe interactions, and microbiota-related disease mechanisms.

The shotgun metagenomic sequencing workflow includes two important steps:

• DNA Fragmentation: The microbial DNA is randomly fragmented into small pieces.
• Sequencing: Universal primers are used to amplify these fragments, which are then sequenced and assembled into longer sequences.

Because this method sequences the entire genome, it can determine not only the genus and species but also, in some cases, subspecies and strains. Additionally, it allows for the analysis of gene expression, function, and how these metabolic functions contribute to community fitness and host-microbe symbiosis.

Choosing the Right Approach

If your research involves sequencing a large number of microbiome samples across different environments or conditions to analyse community diversity, amplicon-based sequencing is likely the best choice. However, if you need to investigate the metabolic and biochemical functions within your microbiome samples, shotgun-based sequencing is the recommended approach. The table below compares some important parameters across both methods:

Cambridge Bioscience has a range of microbiome sequencing services offered by our partners Zymo Research. Contact our genomics specialists today to get started on your project!