Amplicon Sequencing and Metagenomics Overview
by Alberto Acedo on 21/Jul/23
Fishing DNA in the Ocean: Differences between Shotgun Metagenomics vs. Amplicon Sequencing
Imagine a soil sample as a big ocean full of DNA, and a microbial geneticist as a DNA fisherman trying to catch and understand the organisms living in that soil. Just like there are different techniques for fishing in the ocean, scientists have various methods to study the DNA of microbes in the soil.
By understanding these techniques, you can determine which approach is best suited for your research goal. In this article, we will explore the key differences between shotgun metagenomics and amplicon sequencing.
Genetic Molecular Approaches: Trawl Net vs. Fishhook
Shotgun Metagenomics: The DNA Trawl Net
Shotgun metagenomics can be compared to a DNA trawl net that captures and sequences all types of DNA present in a soil sample. It enables the detection of a wide range of organisms, including animals, plants, nematodes, viruses, bacteria, and fungi, all in a single analysis.
Amplicon Sequencing: The Super Fishing Rod with Fishhooks
In contrast, amplicon sequencing solutions can be visualized as both a super fishing rod equipped with millions of fishhooks, each designed to target specific groups of species. This approach allows researchers to selectively fish for the species of interest. For instance, the 16S fishhook is tailored for capturing bacteria and archaea, while the ITS fishhook is specialized for fungi.
By employing these two approaches, scientists can employ the DNA trawl net of shotgun metagenomics to obtain an overview of the genetic content in a sample, or they can utilize the targeted fishhook approach of amplicon sequencing to focus on specific groups of organisms, depending on their research objectives and the depth of information required.
Advantages and Disadvantages
There are various advantages and disadvantages to using one or other methodologies. In this article, we’ll show the basic differences.
1. Species profiling: Trawl Net Size vs. Fishhooks
In the vast expanse of the ocean, a diverse array of species can be found, ranging from whales and dolphins to fish, crabs, clams, and plankton. Similarly, shotgun metagenomics has the capability to capture a range of DNA in a sample, akin to how a trawl net can capture various marine organisms.
However, it is important to note that the notion of capturing "all" DNA may not be your objective. The extent of coverage in shotgun metagenomics depends on the size of the trawl net employed. The size of the net is crucial, as using a net smaller than the size of a whale would prevent the capture of whales altogether. In contrast, this limitation does not apply when using fishhooks. With the appropriate fishhook, it is possible to specifically target and capture a whale if you are a fisherman (or capture a bacteria if you are a microbial geneticist).
As a microbiome specialist, my focus lies in discerning which organisms thrive in which environment based on their interactions and their genetic makeup.
Microbes, in comparison to whales, hold a significantly higher representation in the ocean. While approximately 1.5 million whales are in the ocean, an equivalent number of microbes can be found within just 2 milliliters of ocean water. Due to the abundance of bacteria, when employing a trawl net that cannot cover the entirety of the ocean, the probability of capturing microbes is considerably higher than that of capturing a whale.
In the soil, a similar pattern emerges. Bacterial cells outnumber fungi by a factor of 100 to 10,000. Consequently, when employing shotgun sequencing, the likelihood of sequencing a larger number of bacteria is significantly higher than that of fungi due to this numerical disparity. However, when utilizing amplicon sequencing with an ITS-specific fishhook in metabarcoding, the interference from bacteria is minimized as the target is specifically geared towards capturing fungi. This targeted approach allows for a more accurate and focused analysis of fungal populations without being influenced by the presence of bacteria.
When considering the genomic context, it is crucial to ensure that the Trawl Net size, representing the sequencing depth, is significantly larger than the number of targeted fishhooks or sequencing/DNA reads obtained per sample. Ideally, the Trawl Net size should be at least 100 to 10,000 times greater than the number of fishhooks to achieve comprehensive representation. However, it's important to note that even with a larger trawl net size, the total species representation within a sample may still be limited. Typically, only the most abundant species and those with larger genomes are well represented in the sequencing data.
This can pose a challenge when studying species that play crucial roles in agroecosystems but exist in low numbers. Their limited representation in the sequencing data may obscure their significance and impact within the ecosystem. Therefore, researchers must carefully consider the limitations and biases associated with sequencing depth to ensure a comprehensive understanding of the microbial communities and their functions, particularly when investigating key species that may have a significant influence on agroecosystems.
“Species size” in the genomic context is the genomic size. Multicellular organisms (eukaryotes) are like the big animals in the ocean, their genomic size is 10000-100,000 times bigger than a bacteria genome.
2. Function estimation: Unveiling Ecosystem Roles through Fishing Methods
Both the Trawl Net and Fishhook DNA fishing methods provide data that enables us to assess the potential functionality of the captured species and their roles within the ecosystem.
Enzymes, which drive various biological functions, are encoded in DNA. The Trawl Net approach (shotgun sequencing) allows for the capture of genomic DNA, enabling the complete sequencing of species with high abundance and moderate genome sizes. This comprehensive sequencing facilitates the identification of a significant portion of the enzymatic genetic code.
However, it is important to recognize that the captured genomes represent only a fraction of the species present in the soil or ocean, unless an exceptionally large net is utilized, which can be cost-prohibitive. For the remaining species, only fragments of their genomes are captured, resulting in a variable identification of functions. The extent of function identification depends on factors such as species abundance and genome size.
Not all functions are of equal interest.
Not all functions are of equal interest or relevance in a given context. On one hand, the potential range of enzymatic targets is vast, encompassing thousands of potential enzymes even within the bacterial kingdom alone. Understanding which specific enzymatic targets are of interest in relation to plant growth becomes a crucial factor. However, compared to bacteria, the available knowledge regarding potential enzymes related to plant growth is relatively limited for other taxonomic groups such as plants, animals, and fungi. Consequently, many captured sequences may lack the ability to provide taxonomic or functional information specifically relevant to agriculture.
It is essential not only to identify the total number of functions present but also to assess their representation within a sample. This knowledge allows us to discern the relative abundance of specific functions and link them to corresponding phenotypes, such as increased nitrogen release or enhanced phytohormone production.
Acknowledging that biases stemming from the trawl net size can impact the data obtained is important. This can lead to noise in the form of DNA sequences without utility, resulting in difficulties in identifying low-abundance functions and the potential overrepresentation of certain functions. Therefore, careful consideration and evaluation of these factors is necessary to avoid misinterpretation and ensure a comprehensive understanding of the functional potential within a microbial community.
When it comes to Fishhooks (referring to Amplicon Sequencing), there are two approaches to understanding functionality. One approach involves designing fishhooks to capture specific gene functions, such as enzymes related to nitrogen (N) processes. This approach allows for precise targeting, regardless of species abundance. Another interesting approach is using artificial intelligence (AI) and existing scientific knowledge to identify functions.
Biome Makers revolutionizes soil microbiome analysis with its patented BeCrop Technology, which integrates genomics, microbial networks, and machine learning. Unlike other approaches that concentrate on species-specific metagenomics, BeCrop employs amplicon sequencing, advanced ecological analysis, and machine learning algorithms. By leveraging decades of accumulated research and the largest global taxonomic database, BeCrop predicts and unveils the intricate functions and interactions of the entire soil microbiome. This comprehensive approach offers a deeper understanding of the ecological dynamics at play within the soil microbiome.
To illustrate this, imagine capturing different fish species. By referring to information previously recorded by other fishermen, who have described the functions of those fish species in a book, we can understand their functions. In this analogy, each captured DNA sequence represents a species for which function information already exists. Therefore, if information about the function of a species is available, it remains the same for each individual of that species.
Both the Trawl Net and Fishhooks DNA methods enable the discovery of new species, much like the metagenomic approaches in genomics. For instance, if a new shark species is captured, it may be rare and challenging to identify its complete range of functions using the Trawl Net approach alone. However, with the Fishhook approach, we can observe similarities between the new species and other known shark species that share certain functions. This is where AI plays a crucial role.
3. Cost Efficiency: Fishing Efforts
Both the Trawl Net and Fishhooks methods require varying levels of energy expenditure. Generally, targeted Fishhooks require less energy compared to the Trawl Net method.
There are two main reasons for this:
- Large Trawl Net Required: To capture all represented species and maximize the number of functions in a soil sample, a large trawl net is necessary. This typically requires 100 million to 10 billion DNA sequencing reads, surpassing the capacity of popular fishing boats like the Illumina Miseq, which can handle only 20 million reads. Consequently, more advanced and expensive DNA sequencers like NextSeq or Hiseq are often required. Larger fishing boats also come with higher costs and increased fuel consumption.
- Computational Effort: Effort extends beyond fishing, as classification, comparison, and storage are also necessary. Due to the larger amount and complexity of data generated, the computational effort is typically higher with the Trawl Net method. Dealing with such large datasets requires optimization in data processing, making this a significant consideration
- Energy: The Trawl Net method consumes a significant amount of energy by including non-useful or irrelevant DNA, leading to inefficient energy usage and potential data classification noise.
Considering these factors, both approaches have their own advantages and disadvantages depending on the research question and study design. Researchers should carefully consider the strengths and limitations of each method when selecting the most appropriate approach.
However, if you are a fish lover seeking the best DNA fishing technique, you can rely on the expertise of Biome Makers. Our experts will ensure an ideal fishing experience to provide you with the finest fish that meet your culinary expectations or research interests. We specialize in efficient, balanced, and effortless DNA fishing techniques while maintaining the utmost respect for ocean species. That's exactly what BeCrop offers!
A final note: While soil is important and deserving of our protection, extending the same level of care and concern to our oceans is equally crucial.