As the impact of anthropogenic change on environmental biodiversity becomes an increasingly pressing topic of research, Lab News paraphrases a presentation given by Professor Simon Creer which outlines practical methods of metabarcoding and analysis of eDNA.
Under threat from all sorts of unpleasant, predominantly anthropogenic pressures, biodiversity is important for ecosystem function and services. As well as being an important facet of those individual products we derive from our environment, current research is now investigating biodiversity as a potential indicator of the health of an entire ecosystem.
Requiring vast tracts of data sampling and analysis, traditional, morphological approaches to researching biodiversity on mass is highly challenging both logistically and financially.
So, our understanding of biodiversity and the impact human activities have on our environment is a topic of growing interest and relies on new and emerging technologies.
What is eDNA?
Only a decade ago, the genetic analysis of biodiversity en masse was out of the question. Bulk environmental, or ecological samples contain millions of microscopic organisms from different species and different communities. Spores and pollen can be found in air samples. Macrofauna can be found in terrestrial environments above ground and in soil, and throughout aquatic environments.
Microbes, meiofauna and microfauna can be sampled from subterranean and sedimentary environments. Microorganisms are also found in suspension in aquatic habitats. In order to “count” these organisms using molecular tools, they would need to be carefully separated from the environment, their DNA isolated from each organism and analysed accompanied by eye-watering costs.
Instead, consider the DNA excreted by a stickleback, for example. Free DNA molecules are ubiquitous, released from skin, mucous saliva, sperm, secretions, eggs, faeces, urine, blood, root, leaves, fruit, pollen, and rotting bodies. These are collectively referred to as environmental DNA (eDNA). Rather than collecting DNA from the individual organisms, eDNA is discarded genetic material sampled across airborne, subterranean, and aqueous environments.
How to gather and analyse biodiversity data
To obtain robust biodiversity data, samples must first be collected and preserved. DNA must then be extracted and subjected to some form of molecular biological manipulation prior to bioinformatics, taxonomic assignment of resulting sequences and downstream statistical analysis. Standardisation of these processes can help ensure comparable biodiversity data across studies.
There are several methods for looking at biodiversity from a molecular biological perspective. Quantitative polymerase chain reaction (qPCR) represents high standards in both qualitative and quantitative assessment of biomass but tends to be used for species-specific research as it is both labour intensive and very expensive. It is also possible to employ high throughput metagenomic methods to sequence the collective genomes of everything in the sample using shotgun environmental sequencing.
However, for research that aims to identify multiple species from a multiple, independent samples, metabarcoding (also known as mixedtemplate PCR, amplicon-sequencing, or markergene analysis) employs general or universal PCR primers to amplify taxonomy marker genes across multiple species followed by high-throughput sequencing analysis to generate millions of barcode markers - short, harmonised fragments of DNA from the same location on the genome co-located in organismal genomes.
Similar to all high throughput methods, metabarcoding generates thousands to millions of reads which can be analysed on a computer. So, it has only been in the last decade or so that this method has become accessible, through the increasing power of computers, to a broad research audience.
Getting eDNA biodiversity analysis right
Defining the target community is essential to all biodiversity studies. Selection of the correct primer to ensure the target taxa are amplified and choosing the correct taxonomic marker – eukaryotes, generic vertebrate, plant, or insect - will also help determine whether or not you can resolve species within your chosen community in the analysis.
It is also possible to carry out a two-step PCR, adding a small section of DNA onto the end of each sequencing read, for different samples. This enables our computers to target only those sequences and collate and cross-reference them to integrate information across many samples. It is also important to understand how complete the reference database is for the selected gene taxonomy markers.
eDNA is discarded genetic material sampled across airborne, subterranean, and aqueous environments
Evolutionary relationships between samples can be reconstructed using phylogenetic inference. This identifies homologous characters across a data set before comparing them to a reference database that reconstructs the evolutionary tree as it is currently understood. While high throughput NGS improves our understanding of taxonomic details for more and more families of organisms, there remain gaps in these databases.
Although an incomplete database doesn’t necessarily halt an analysis, it will determine the resources required to complete it and the taxonomic level at which your analysis can take place. For example, if no species from the same genus are available in the database, you may only be able to assign a taxonomic family to that sequencing read derived from the unknown species.
The pros and cons of eDNA metabarcoding
Metabarcoding datasets are compositional, such that species counts are given an ‘arbitrary total imposed by the instrument’ and this bias should be accounted for in analytical studies . While these studies cannot determine population quality data such as sex ratios or species conditions and should only be recognised as semi-quantitative, they can detect rare or invasive species and changes in biological communities following human intervention. Varying degradation of eDNA due to high temperatures and humidity and movement through media such as water can affect spatiotemporal data trends. A rotting fish on the edge of a river, for example, will release eDNA that can be detectable up to a kilometre and a half downstream.
Datasets are being used to successfully evidence the presence of rare species such as the Great crested newts (Triturus cristatus) , map the textural heterogeneity of soils to microbial and fungal diversity , reveal the annual life-cycle of key indicator species to monitor entire ecosystems , monitor shifts in honeybee foraging impacted by changes in floral resources, and specify the allergenic potential of different grass pollens throughout the year .
With the potential to contain the life histories of every species associated with a specific area, eDNA analyses offer vast amounts of biodiversity information. Studies of eDNA are non-invasive, can target specific species, sample greater diversity, and improve taxonomic resolution. Data gathered is ecologically relevant to what is happening in the real world opening the door to a future of biodiversity discovery and ecological understanding at scales that have previously been impossible.
- Lab News has paraphrased a recent presentation by Professor Simon Creer which outlined practical methods of metabarcoding and analysis of eDNA. Professor Simon Creer, Molecular Ecology and Evolution, Bangor University, mefgl.bangor.ac.uk/index.php.en
- Kelly et al. 2019. Sci Rep 9, 12133.
- Buxton et al. 2022. Sci Rep 12, 1295
- Seaton et al. 2020. Soil Biol. Biochem.
- Bista et al. 2017. Nat. Comms.
- George et al. 2019. Nat. Comms.
- Jones et al. 2021. Comms. Biology
- Brennan et al. 2019, Nat. Ecol, Evol
- Rowney, Brennan et al. 2021. Current Biology