eDNA and other monitoring methods based on molecular biology

With the help of molecular biology, we can improve the coverage, accuracy, and cost-effectiveness of environmental monitoring, enabling a much more complete picture of biodiversity and the state of the environment and their trends.

On this page:

• What are molecular monitoring methods?
• Most commonly used molecular methods
• Possibilities and challenges of the methods
• Standardization and quality control
• Implementation in Finland
• How to implement molecular methods?
• Welcome to the national eDNA idea group
• Resources

What are molecular monitoring methods?

Molecular methods refer to techniques based on DNA or RNA analysis, which can be used in nature monitoring. Based on DNA, a single target species can be detected or even hundreds of species can be identified from individual samples. Population structure and other intra-species variation can also be studied using DNA methods. RNA-based methods produce information about the genes expressed in the studied environment and thus about the biological processes going on in the ecosystem.

Many types of samples can serve as source material for molecular analyses. To verify species identification and to study genetic variation within the species, tissue samples taken directly from the organism, or for example, excrement samples are most often used.

Environmental DNA samples are usually used to either identify a specific target species or to find out the species of a certain species group or groups as comprehensively as possible. Also for samples collected with traditional sampling methods, such as fishing, the species can be identified with eDNA methods instead of morphological identification. This is done from a DNA homogenized compilation or a bulk sample or for example, from the liquid used to preserve the sample.

The costs of massive parallel sequencing and other methods, which enable the simultaneous detection of even hundreds of species, have significantly decreased in recent years. Chains of methods, from sampling to interpretation of results, have developed to a degree that enables their large-scale, routine use in environmental monitoring. With the help of new methods, the coverage, accuracy, and cost-effectiveness of monitoring can be improved enabling a much more complete picture of biodiversity and the state of the environment and their trends.

Most commonly used molecular methods

Ensuring species identification of an organism: DNA barcoding

In DNA barcoding, DNA is isolated from a sample taken from an organism and a certain short region suitable for species identification (“barcode”) is sequenced, whose base sequence is compared to a database of confirmed species-specific barcode sequences. In addition to traditional Sanger sequencing, massive parallel sequencing (so-called second and third generation sequencing) is also used in DNA barcoding today.

Identification and abundance determination of individual target species: PCR tests, qPCR, dPCR, ddPCR

In a PCR test only the DNA of the target species is duplicated from the DNA isolated from the sample using a species-specific primer. With quantitative PCR methods (qPCR, dPCR, ddPCR) it is also possible to determine the abundance of DNA of the target species in the sample.

Species determination: metabarcoding (2nd and 3rd generation sequencing)

In metabarcoding, up to hundreds of thousands of sequences of a certain barcode area are produced from one environmental sample by massive parallel sequencing. The sequences can be identified by comparing the DNA base order with the sequence database. The target group of organisms is limited by the barcode area and the PCR primers used in DNA duplication. With metabarcoding, it is possible to find out a large part of the species in that species group in the sample. In addition, the method provides indications of species abundance ratios. The proportion of identified species and the accuracy and reliability of the identifications primarily depends on the quality and comprehensiveness of the reference database used.

In metabarcoding, the so-called second-generation sequencing technology (e.g. Illumina NovaSeq, MiSeq, NextSeq), which requires DNA duplication and sequencing to be limited to a specific gene region of a maximum length of approx. 300 base pairs. The newer so-called third-generation sequencing technology (e.g. Oxford Nanopore, PacBio) can produce much longer sequences (tens or hundreds of thousands of base pairs) from the entire genome area, and DNA duplication is not necessary.

In principle, the new technology makes it possible to reach all groups of organisms with the same analysis and also to examine intra-species variation. In practice, however, the utilization of the method is still limited by the coverage of whole genomes and genome libraries, the fragmentation of DNA obtained from environmental samples into smaller pieces, and to some extent also the insufficient quality of long sequences.

Exploring intra-species variation: sequencing of single genes, genomics, SNP and microsatellite marker genes

With current methods, the investigation of intra-species variation usually requires samples that can be linked either to a single individual or to a specific population. Determining intra-species variation from eDNA samples collected from the environment has been developed in a few research projects, but suitable methods are still unattainable for most species for the time being.

Genetic diversity within a species can be assessed with many different methods, which aim to answer slightly different questions. They also differ in how much background information about the genetic structure of an organism must be available in order for the method to be applied. However, with the decrease in sequencing costs and the development of methods, the collection of genetic background information is no longer a major bottleneck for examining the intraspecific genetic diversity of natural populations.

Possibilities and challenges of the methods

eDNA and other molecular methods enable species identification that is easily comparable and reproducible internationally. With their help, it is also possible to reach many difficult-to-detect and poorly known groups of organisms that are outside the scope of monitoring based on traditional methods. For example, local water and air samples can be used to reach either individual species or the entire species in a large area, which would not be possible to map with traditional methods due to costs.

Although molecular methods can significantly supplement the overall picture of biodiversity, they do not produce information comparable to methods based on traditional species identification. For example, meta-barcoding does not provide information on the number of individuals of a species or the characteristics of individuals such as age, size or sex. In addition, the information about the relative abundance ratios of species obtained by meta-barcoding is still generally only indicative at the moment. However, methods are constantly being developed in a more quantitative direction.

Another significant challenge is related to the interpretation of environmental DNA data. Since environmental DNA detects DNA present in for example water and not directly living individuals, it is necessary to evaluate on a case-by-case basis what the observations tell about the presence of individuals. The transport of DNA and the rate of degradation in the environment are affected by numerous factors, such as flow conditions, temperature, and sunlight. The degradation and migration of DNA in different environments has been investigated in many scientific studies, the results of which should be taken into account when planning molecular biological monitoring. Various models describing the migration of DNA are also useful in evaluating possible source areas, but the predictions of the models are inevitably always accompanied by uncertainty.

Molecular monitoring methods are currently being actively developed around the world for different groups of organisms and ecosystems, and individual methods have also been put into routine use in several countries. However, the use of molecular methods in monitoring on a large scale is still at an experimental stage, and the field of development projects and expertise is fragmented.

Insufficient funding, lack of skilled workers, gaps in reference libraries especially for certain northern species groups, and the lack of international standards for methods are considered by experts in the field to be the most important factors limiting the introduction of methods. There are national and international methodological guidelines, but they are scattered, and the minimum requirements have not been generally accepted or agreed upon, i.e. there are hardly any real, commonly accepted standards yet. Decentralized funding easily causes guidelines to be developed at the national or organizational level, but there is not enough exchange of information or sharing of lessons learned between projects and different countries. This causes a great risk that the molecular biological data are not comparable at the international level.

Standardization and quality control

International standardization of methods is very important to ensure the quality and comparability of molecular biological data. Standardization is currently still in its early stages, but it is being actively promoted by many different measures in Finland and internationally.

The CEN standard EN 17805:2023 Water quality – Sampling, Capture, and preservation of environmental DNA from water has been published in early 2023. An eDNA-themed working group is being established under ISO/TC 147/SC 5/WG13 “Environmental DNA and RNA methods”. Development manager Kristian Meissner from the Finnish Environmental Insitute oversees the working groups for the already published CEN standard and the ISO standard that is currently under preparation.

The International eDNA Standardization Task Force iESTF (https://iestf.global) was established in connection with the international GEO BON conference in Montreal in October 2023. iESTF cooperates closely with the international research community and various stakeholders.

Implementation in Finland

Molecular monitoring methods are still mostly in the experimental stage in Finland, but the development is fast. In addition to universities, method development and piloting are carried out in all key institutions that coordinate environmental monitoring (Syke, Luke, Central Museum of Natural Sciences, Forestry Agency, Meteorological Institute, Health and Welfare Institute, Food Agency).

The methods are in routine use only for individual game and fish species. The monitoring of entire communities of species using metabarcoding methods has not yet been put into routine use in Finland, but large-scale piloting has been done or is underway, especially with aquatic organisms such as plankton and epiphytic algae, benthic animals, and fish. In the terrestrial environment, large-scale monitoring based on metabarcoding is being piloted, especially with arthropods and fungi.

In Finland, efforts are being made to promote the introduction of molecular monitoring methods, e.g. with research funding and coordination and cooperation between different actors. Molecular methods are included in the Environmental State Monitoring Strategy 2030 published by the Ministry of the Environment, and Syke and Luke have drawn up a road map for the introduction of molecular monitoring methods.

Table: Molecular monitoring schemes in Finland.

How to implement molecular methods?

We have compiled answers to concrete questions that arise for those interested in the use of molecular monitoring methods. If you can’t find an answer to the question that is bothering you, please contact us and we will try to help.

Resources

More information

Veera Norros
Tiina Laamanen
firstname.lastname@syke.fi