Welcome to the exciting world of metagenomics! Metagenomics is a powerful tool that allows us to study complex microbial communities in their natural environments.
It has revolutionized our understanding of how microbes interact with each other and their environment, and it can help us better understand many aspects of biology, from disease diagnosis to climate change.
In this article, we’ll take a look at some of the most popular metagenomics techniques and explore how they are used in research today. So let’s dive in and discover what metagenomics can do for us!
Intro to Metagenomics
First things first, we need to cover some basics about metagenomic analysis and what it is.
What is metagenomics?
Metagenomics is a term used to refer to whole genome sequencing analysis of microbiome samples. The microbiome consists of the community of microorganisms found in a sample and their genetic content.
These complex microbial communities are present in various sample types, including on and in bodies (human, animal, fish, insect, etc), within soil, water, and air samples, and even in the built environment (building walls, subway walls, within HVAC systems, in sewers, etc) and food.
Metagenomics is a group of technologies, tools and approaches that allow the direct genetic analysis of the total genetic content contained within a sample. This functional analysis allows researchers to determine the identity, abundance and activities of microorganisms present and provides insight into a sample’s microbial diversity.
This can aid in gene prediction, provide a better understanding of microbial communities and how they interact with their environment as well as uncover new genes, pathways and organisms.
What are the core applications of metagenomics?
Metagenomics has many applications in the medical field, such as the identification of pathogenic microorganisms in the intestinal tract, bloodstream infections, lung infections, central nervous system infections, and other infections.
Applications of environmental metagenomics include the study of microbial species in oceans, soils, deep oceans, glaciers, craters, and other environmental samples.
At present, some metagenomic research focuses on antibiotic-resistant bacteria (ARB) and antibiotic-resistant genes (ARGs), biocatalysts, drugs, and others.
While some studies do look at antibiotic resistance genes, not all do. Many studies still focus on identifying the microbial community composition of bacteria present and how they change with disease, treatment, environment, time, etc. It is largely recognized that the functions of these communities are more important than just who is there.
Additional applications of metagenomics include:
- Food pathogens and food production workflows as fermentation may be surveilled using metagenomics.
- Metagenomics helps to understand the microbial diversity and dynamics of captive animals in conservation and improve their welfare and fitness.
- Pets could be monitored to evaluate dietary impact using metagenomics.
- Metagenomics could be applied to monitor microbial DNA, resistance and virulence dynamics in farm animals.
What are the advantages & disadvantages of metagenomics techniques?
Metagenomic techniques have a number of advantages and potential challenges that are important to be aware of.
Advantages:
- Detection of unculturable microorganisms through genomic assemblies
- Identification of a broad range of microorganisms present in a sample without the need for laborious culturing methods with strain level accuracy
- High-throughput of samples
- Identification of genes, allowing prediction of functional potential
- Multi-kingdom identification of (bacteria, protists, archaea, fungi and viruses).
Disadvantages:
- The identification of microorganisms is database-dependent.
- Results in large data sets can be complex and difficult to analyze.
- Requires specialized knowledge of analysis techniques and statistics.
- Validation by experimental methods is recommended to confirm the findings.
- The assembly success of genomes and accuracy of output genetic sequences are metagenomics technique-dependent and are often traded off for one another.
Understanding Metagenomic Techniques
To help you understand the steps and process of metagenomic techniques, here’s our step-by-step guide:
Sample collection
The gold standard practice here is to collect a sample and immediately snap-freeze it in liquid nitrogen and transfer it to -80C for long-term storage. This provides the most accurate snapshot of the microbial community
Another option is to place the sample in a preservation buffer, like Zymo’s DNA/RNA Shield, that preserves the integrity of the nucleic acids and makes the sample stable at room temperature.
DNA extraction
After sample collection, it’s time for DNA extraction. The best method for bacterial cell walls involves a combination of physical lysis (e.g. bead beating, sonification) and chemical lysis (e.g. lysozyme). This ensures that both Gram-negative and Gram-positive cell walls are broken down for efficient DNA extraction.
Some sample types, such as soil, require the removal of PCR inhibitors and humic acid to ensure successful PCR amplification.
If you suspect your samples have humic acid or other substances that may hinder PCR, it’s best to use a DNA extraction method that is specifically designed for environmental samples.
Library preparation
Following DNA extraction, the sequencing library preparation is performed. This involves adding indices to individual samples, fragmenting the genomic DNA, amplifying the DNA, and pooling the samples.
By pooling multiple samples, you can reduce the cost of sequencing. Additionally, this allows for the identification of shared taxa and differential abundance calculations between samples. After library preparation, the samples are ready to be sequenced.
Sequencing
Illumina next-generation sequencing is performed on the pooled libraries. This employs short-read sequencing-by-synthesis technology which is widely used for metagenomic sequencing. The data is then transferred to a bioinformatics pipeline for analysis.
Data Analysis
After sequencing, raw reads are processed through a bioinformatics pipeline. This is to quantify DNA sequences that match unique microorganisms to profile the microorganisms present and their relative abundances.
These pipelines may be assembly-based, and assemble reads into contigs prior to mapping them to taxonomy or read-based, and map reads to genomes or other marker genes. Functional genes may also be identified from this data.
Once taxa and functions have been identified and their relative abundances calculated, statistical analysis can be performed. This includes the calculation of diversity indices (alpha and beta diversity) to identify overall species richness, evenness and community composition trends in the data along with analyses of individual taxa or functions.
Differential abundance analysis could be performed to test if individual taxa or functions are enriched in a sample.
Key Metagenomic Sequencing Techniques & Their Uses
Targeted metagenomic sequencing (i.e. 16S rRNA gene amplicon sequencing)
Through amplicon sequencing, researchers can focus on a specific region of the genome in order to reveal its unique contents.
Amplicon sequencing, specifically 16S rRNA gene sequencing, is an incredibly valuable tool for identifying bacteria and archaea. This type of sequencing relies on amplification of the same conserved region of the 16S rRNA gene (shown below), which allows for detection of all bacteria present in the sample. Within these conserved regions are variable regions unique to individual bacterial species, making it possible to identify taxonomic differences within communities.
Moreover, the variable regions in the preserved 16S rRNA gene enable us to analyze the genetic distance of organisms present in a sample, recognize microbes and deduce their relative amounts.
Amplicon sequencing enables the identification of a variety of microbes, such as fungi through ITS sequencing and eukaryotes like protists by means of 18S sequencing.
Figure 1: The 16S rRNA gene contains conserved regions that can be used to target primers which amplify the variable regions, which in turn can be used to identify the microbes present. Some common primer sets are shown below the gene.
Shotgun sequencing
Shotgun metagenomic sequencing is a method that involves randomly breaking DNA into small, piece-like fragments akin to what happens when something is blasted with a shotgun. Unlike amplicon sequencing, there is no targeting of a specific segment of the genome, which means the entire genetic content of the sample gets sequenced.
This avoids bias introduced by primer binding and amplification and allows for the additional identification of functional genes and pathways that are not detectable by amplicon sequencing.
The most common shotgun sequencing method used is sequencing-by-sythnesis (SBS). During SBS, the short DNA fragments are bound to a flow cell. Fluorescently labeled dinucleotides, namely adenine (A), thymine (T), guanine (G) and cytosine (C), are then added to the mix.
The bases bind in a complementary manner to the template DNA strand and the fluorescent dye is then cleaved off. The fluorescent signal is read sequentially, allowing determination of the DNA sequence. This process is done in a massively parallel manner, allowing for quick turn-around times.
Using bioinformatics, the sequence reads acquired from a sample are then linked to reference databases or crafted into microbial genomes in order to recognize which species and genes exist.
Final Thoughts
For researchers who think their study design does not require cross-kingdom strain level resolution, 16S rRNA amplicon sequencing may be enough. However, for studies to inspect cross-kingdom species at a strain level and their functional genomics, the go-to option would be shotgun metagenomcs sequencing.
Whatever your sequencing needs, CosmosID has state-of-the-art tools to support you.
At CosmosID, our microbiome sequencing services and industry-leading bioinformatics capabilities provide customers with unprecedented insight into the microbial ecology of their samples.
Our suite of services allows for rapid, cost-effective identification and analysis of microorganisms in environmental and clinical samples to meet any research need. For more information on our metagenomic sequencing services, please contact us today!
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Metagenomics Techniques FAQs
What techniques are used in metagenomics?
16S and 18S rRNA and ITS amplicon sequencing and whole genome shotgun sequencing are common metagenomics sequencing methods.
What are the main steps in a metagenomics study?
From sampling and storage to extraction, library Prep and sequencing, followed by bioinformatics analysis – the process of studying biological data is a multi-step approach requiring precision and meticulousness.
What are the principles of metagenomics?
Metagenomics is based on the principle that the communities of microbes and their genes in microbiome samples can be identified by mapping DNA sequences to databases of known features.
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