Module 1 Genomics: environmental DNA and sampling

1. INTRODUCTION

Sampling, extraction, and analysis of DNA persisting in the environment is one of the most essential technological and scientific innovations of the last decade. Retrieving DNA from environmental samples, the so-called environmental DNA (eDNA), is a powerful approach for obtaining information about species, populations, and communities regardless of their physical presence in nature as individuals or as culturable microorganisms. The persistence of DNA in the environment and the fact that it can be sampled, extracted, and analysed are considered essential scientific and technological advancements of our times related to species monitoring and inventory of their natural habitats. Thus, eDNA sheds light on fundamental and applied research in molecular biology, environmental science, ecology, palaeontology, etc.

Using eDNA from a single sample, one can simultaneously analyze the DNA from an entire microbial community comprising different taxonomic categories. The analysis consists of PCR amplification of the eDNA followed by DNA sequencing. The amplification is arranged through a single-species or multi-species approach that exploits species-specific and generic primers for a given target group of organisms.

The high throughput technology of Next-Generation Sequencing (NGS) is rapidly advancing nowadays and contributes to the performance of comprehensive surveys grounded on the multi-species eDNA approach. Since this technology application is expanding, it offers a substantial cost-to-value advantage compared to the classical sequencing methods. Furthermore, the multiple-species eDNA approach is gaining progressive popularity due to the DNA metabarcoding technique development. DNA metabarcoding stands for the performance of mass DNA sequencing for simultaneous identification at the molecular level of multiple taxa in an environmental sample. The target sequences amplified by PCR, sequenced, and applied as barcodes to find discriminative taxons, are most commonly those of genes that are highly informative at the species level and present in a high copy number.

The application of eDNA approaches for describing organisms’ biodiversity of ancient and contemporary ecosystems through obtaining genetic material directly from the environment is a background on which various molecular methodologies and analytical tools can be applied to reveal the full potential of the molecular biology branch of environmental genomics.

2. GENOMICS AT A GLANCE

Genomics is a bioscience that analyzes the interactions of genes as biological information objects at species, population, and ecosystem levels. It deals with vast sets of genetic information and analyzes it automatically through network concepts with the help of high-performance computing and bioinformatics technologies.

As a DNA-based approach for genome data generation and interpretation, genomics’ roots go back to the 1970s when Fred Sanger’s group developed a gene sequencing method and using it completed the first three genomes, those of the bacteriophage Φ-X174, the genome of the human mitochondrion, and the genome of the λ virus. Two years later, Walter Fiers’ team determined for the first time the sequence of a gene – the bacteriophage MS2 coat protein gene. During the same decade, the Φ-X174 bacteriophage genome was the first DNA genome completely sequenced, and almost 20 years later, in 1995, the first free-living organism genome was sequenced – that of the bacteria Hemophilus influenzae. Since then, genomes of species belonging to the three domains of life – Archaea, Bacteria, and Eukarya have been sequenced at a tremendous pace.

Currently, genomics is further differentiated into several subtypes depending on the level it operates and the gene identification approaches it uses.

• Functional genomics. It deals with the analysis of genes at the functional level. The methodological approaches include two genetic approaches, complementary to each other:
  • Forward (classical) genetic methodology that encompasses randomly generated mutants of the desired phenotype and exploits them in finding the normal gene sequence and its function;
  • Reverse genetic methodology uses the normal gene sequence (obtained by genomics) and induces targeted mutation in that gene to observe how this mutation alters the phenotype. Based on this alteration, the function of the normal gene is deduced.
• Microarray genomics. It enhances the identification of a complete subset of genes that operate during specific temporal or developmental stages of organism growth. The total set of genes transcribed during an event of interest (e.g., microbial growth in the presence/absence of O₂, root hair growth in a plant, or limb bud production in an animal) can be determined through this approach. Although the forward genetics objective is the same (an assemblage of the genes subset referred to a specific biological process or conditions), this technique identifies genes for which mutations are induced, and their phenotyping effect is easily traceable.
• Comparative genomics. It studies the evolutionary relationships among organisms and contributes to our knowledge of life phylogeny. Methodologically, with the sequencing of entire genomes, comparative genetics offers the detection of much more specific and minor differences in the organisms and thus, complements the data of the classical genetics (chromosome size and number and banding patterns among populations, genera, and species) for the evolutionary relationships. The main advantage of comparative genomics in this sense is that it compares the DNAs of organisms directly on a small scale. Since the DNA sequences are processed and measured through a mathematics approach, the genomic analysis is precisely quantified and can measure specific degrees of relatedness.
• Environmental genomics. It integrates environmental data and metadata accumulated at various levels of organisms’ organization (from cell to ecosystem). This branch of science favors interdisciplinarity as a philosophy and methodology in understanding the complexity and functions of genes and organisms with their dynamics and evolution in temporal and spatial contexts. It aims to characterize at a genetic level the factors that contribute to the diversity of organism responses to environmental stressors. One of the most powerful tools of environmental genomics is the identification and study of environmentally responsive genes across the species to accelerate the discoveries of environmental health science.

3. ENVIRONMENTAL DNA

3.1. Definition – eDNA as a molecular monitoring tool 

eDNA, short for environmental DNA, is organismal nuclear or mitochondrial DNA released in the environment. eDNA may originate from any cellular material. Typical sources of eDNA from macroorganisms are shed skin and hair, urine and secreted excrements, carcasses, mucous, gametes, and dead individuals. eDNA from microorganisms (both pro- and eukaryotes) is derived from a whole organism. eDNA can be found anywhere in terrestrial (soil) or aquatic (water and sediment) habitats. It can be sampled, detected, and monitored via molecular biology techniques regardless of the presence of any biological source material. eDNA persistence in nature is mainly temperature-sensitive. It can be preserved for a few weeks in temperate water up to hundreds or thousands of years in ever-frozen habitats. In aquatic habitats, eDNA concentration is very low and easily dispersed due to the hydrological processes. In terrestrial habitats, eDNA samples are enriched in the target material compared to the aquatic ones. Regardless of the habitat, eDNA exposure to UV radiation, elevated temperature, low pH, and endo- or exonucleases can lead to its rapid degradation.

Historically, eDNA flourished with the research idea to obtain nucleic acids of microorganisms directly from the environment in the late 80s of the XX century. It was the first approach to gain the true picture of microbial abundance in nature since the culture-based methods for studying microbial diversity misrepresent the situation in natural conditions. eDNA was considered the unique tool revealing their genetic representation and bringing insight into microbial diversity and functional genomics.

Nowadays, eDNA is used as a tool for the detection of targeted species and assessment of biodiversity in ecosystems. It can be defined as an approach for obtaining genetic material from the environment followed by a diversity of molecular methods and tools used for analysis to gain deeper insights into fundamental and applied aspects of species inventorying and monitoring, ecology, conservation biology, paleontology, and environmental science.

3.2. eDNA application areas

3.2.1 eDNA of microbial origin 

eDNA metabarcoding, as an emerging method for biodiversity assessment, comprises taking samples from the environment (aquatic, terrestrial, or even air), extracting DNA, amplifying it through PCR with general or universal primers, and sequencing the amplification product through NGS to generate a huge amount of raw sequencing data. These data are the source of valuable information about the presence of defined species in a community and assessment/monitoring of its biodiversity across all habitats and taxonomic groups. Thus, eDNA metabarcoding is an essential tool for ecological monitoring and conservation studies.

eDNA metabarcoding as a methodological approach is characterized by high sensitivity, taxonomic selectivity, and cost-efficiency, comprising an efficient and precise biomonitoring tool. For almost a decade, eDNA metabarcoding has been exploited for the characterization of aquatic and terrestrial habitats, among which that are hard to reach by traditional eco-monitoring tools, not only for species composition but also for detection of bio-invasion, studying the ecology of trophic chains, identification of cryptic, rare, or threatened species, reconstruction of ancient ecosystems, monitoring of air, water, and soil quality.

Terrestrial habitats

Despite the methodological challenge of the short persistence of eDNA within the soil, eDNA metabarcoding is used for species detection in terrestrial ecosystems. eDNA is applied for the terrestrial ecosystems’ microbial communities characterization with contributions to their structure and function study. Functional annotation of the predicted proteins from different terrestrial habitats and their assignment to various orthologous groups provides for estimation of the microbial diversity abundance or scarcity in these very habitats. Such functional analysis, combined with two-dimensional cluster analysis, allows for a comparative study of the functional profiles in terms of encoded proteins of related terrestrial habitats and to find which genes are particularly enriched or restricted to a given habitat. All this allows the researchers to predict functional differences among the microbial communities. Furthermore, this approach provides for understanding genotype-phenotype relations and building ecosystem dynamics models that shed light on global microbial biodiversity. eDNA metabarcoding reduces the costs for microorganisms’ sampling, overcoming the hurdle of the culture-dependent methods for microbial species abundance studying and the negative impacts thereof.

Applying the shotgun sequencing of eDNA, isolated from various terrestrial habitats, combined with quantitative gene content analysis, resulted in habitat-specific microbial fingerprints generation. These fingerprints are attributed to peculiar sample environments and reflect its specific characteristics.

On the other hand, gene identification, typical for a given environment through gene-centric comparative analyses, reveals new prospects for interpreting and diagnosing environmental data. Thus, eDNA metabarcoding of samples from microbial pathogen-endemic regions can be used to detect the presence of pathogens and their potential host organisms together with microbiomes associated with the persistence of these pathogens in the environment. Moreover, eDNA metabarcoding can be applied to monitor and surveillance of pathogens. It can be used for systems’ design to monitor host-pathogen-environment interaction and early warning of disease risk before disease manifestation.

The facility and universality of the application of eDNA metabarcoding are  used to observe and study endangered habitats on Earth by detecting invasive species and conservation of the endangered ones.

Aquatic habitats

The relatively fast dispersion and low concentration of aquatic eDNA make the traditional methods for microbial species assessment in aquatic samples a real challenge. However, measuring microbial communities within fresh and marine water by eDNA metabarcoding through the generation of thousands of amplicon sequences helped detect native (small or rare) species of certain aquatic habitats and improve biodiversity assessment. eDNA barcoding helps quantify both pro- and eukaryotic microbial lifeforms and detect extant species, describe new ones, and improve the overall understanding of the complex microbial dynamics of aquatic ecosystems. In addition, eDNA metabarcoding may be used for invasive aquatic species determination. This is done by applying periodic screening procedures for the simultaneous detection and monitoring of various invasive species.

A profound study of the bacterial rRNA amplicons of samples that encompass the aquatic habitats of the global ocean – from the surface to the deep-sea floor revealed the differences between pelagic and benthic microbial communities at all taxonomic levels. The microbial communities’ composition and dynamics were compared in surface- and deep-waters and oxygenic/anoxygenic ecosystems. The established differences reflect the heterogeneity and dynamics of these habitats and the environmental restrictions (e.g., availability/lack of oxygen, land influence, surface water productivity, water quality, etc.) there. All these data indicate that the global bacterial distribution study across aquatic ecosystems brings for horizontal and vertical mapping of microbial communities, drawing the regional picture of microbial diversity, and launching distribution patterns that define the biogeographical bacterial biomes in the aquatic ecosystems.

3.2.2 eDNA of macro-organisms

eDNA extracted from macroorganisms differs in nature from the eDNA of the microorganisms. While the microbial eDNA represents the whole living organisms in a sample, the eDNA from a microorganism in a sample corresponds only to a part of organism-free DNA and cellular remains.

The discovery and study of macroorganisms’ eDNA is appropriate for biodiversity conservation purposes and is applied to different terrestrial and aquatic environments, both modern and ancient. Besides the sample nature, the workflow of eDNA studies encompasses the following basic steps (Fig. 1.1.):

• Environment (modern or ancient) selection;
• Sampling;
• DNA extraction along procedure specific to the relevant environmental sample;
• PCR amplification with the aid of generic/species-specific primers to reveal biodiversity;
• Amplicon sequencing;
• Bioinformatic data processing;
• Species identification and results interpretation.

 Biodiversity assessment in ancient environments

The ancient environments comprise terrestrial and aquatic sediments. Namely, these sediments were the first from which eDNA was extracted and studied for macroorganisms biodiversity assessment. The achievements of the eDNA approach in ancient ecosystems contribute to describing past biodiversity and shed light on macroorganisms’ evolution history. Moreover, the eDNA of ancient environments can change the time frame of population demography and contribute to making extinction predictions more reliable. These more reliable considerations, in turn, can help for better conservation strategies’ design and implementation. eDNA from skin cells, feces, or urine can serve as a support proof to the last-appearance dates for extinct species establishment.

The main achievements of these studies are summarized in Table 1.1.

Figure 1.1. eDNA studies flowchart. (Source: Thomsen & Willerslev, 2015)

Table 1.1. Biodiversity assessment in ancient environments.

Type of eDNA	Samples/Characteristics	Applications
Sediments
Terrestrial sediments
– Sedimentary ancient DNA (sedaDNA)	– Permafrost samples of local origin – Derived from faeces, urine, epidermal cells and hair of organisms’ (mammals, invertebrates, birds, plants) remains present within the sample	– Reconstruction of paleo-ecosystems and demonstration of species richness of ancient plant and animal communities
– DNA leaching through strata	– Between strata of non-frozen deposits
Aquatic sediments
– Marine eDNA	– The largest reservoir of eDNA in the oceans – Anoxygenic conditions preserved the eDNA reducing nuclease-driven degradation	– Applied for assessment of anthropogenic impact on marine ecosystems; – Use of NGS for revealing of species diversity in microgeographic regions
– Estuarine eDNA	– Useful for comparison of eukaryotes assemblages
– Fresh-water eDNA	– Proved matching between the fresh-water sediments species (based on eDNA of plant origin) taxonomic representation and that of the relevant macrofossils	– Complementing analyses of macrofossils and pollens for better biodiversity revealing – Better insight in the ecological and evolutionary consequences of environmental changes
Glaciers
– eDNA	– Ancient ice cores of local and distant origin	– Provides insights into the past ecosystems’ plants and animals

Biodiversity assessment in modern environments

The biodiversity assessment in modern ecosystems is crucial for understanding the complex species assemblages’ structure and function. It helps measure the ecosystems’ health and dynamics in normal and under environmental fluctuations and homeostasis disturbance.

The main approaches and results of macroorganism’s biodiversity assessment in various environments are summarized in Table 1.2.

Table 1.2. Biodiversity assessment in modern environments.

Type of eDNA	Samples/Characteristics	Applications
Terrestrial habitats
Surface soil
– Intra/extracellular eDNA	– eDNA metabarcoding	– Reveals species communities’ richness and diversity – Exploits bioindicator groups of ecosystem health, identified by NGS – Reflects overall taxonomic richness and relative biomass of individual species
Aquatic habitats
Fresh water
– eDNA from macro-organisms	– Fast eDNA decay proves for contemporary presence of species and populations	– Application to conservation approaches – Detection of invasive species – Presence of low-abundance species – Monitoring of endangered species (estimation of relative abundance) – Quantification of biomass and species composition
Sea water
– eDNA from macro-organisms	– eDNA metabarcoding	– Monitoring and management of marine biodiversity and resources

4. eDNA SAMPLING 

 4.1. eDNA protocols development

Sampling

Regardless of the targeted environment (terrestrial or aquatic, ancient or contemporary), the first step in the eDNA workflow is the sampling.

Water sampling: collection and preservation (with ethanol or Na-acetate) of water samples directly from the source or after filtration through filters of various materials (cellulose-nitrate, glass fiber, or carbonate). The direct sample requires immediate freezing, and the filter-based ones – freezing or dehydration of the filter matrix with ethanol.

Soil sampling: surface soil samples are collected and sealed in polyethylene sterile bags and stored at 4 ^oC for processing before freezing at -80 ^oC. Alongside, biochemical analyses of soil samples from the same site are performed to provide data about their chemical composition and characteristics (organic matter presence, essential and nonessential elements), and organisms present (microscopical observation).

DNA extraction

eDNA is isolated following well-known state-of-the-art protocols and kits considering the relevant environmental sample. Once extracted from the samples, the eDNA is stable if purified and preserved by freezing. An important issue that can jeopardize the result, is the putative cross-contamination between species. That is why caution is needed in the sample storage and eDNA extraction procedures.

PCR amplification

Although eDNA can be analyzed by conventional PCR methods, quantitative PCR (qPCR) is the preferred tool since they are more sensitive, does not cross-amplify, and does not give false positive reactions. The crucial step in qPCR is the primers and probe design.

The primers are designed to satisfy the two distinct approaches: the single-species approach targeting a species-specific DNA region and the multispecies approach that uses generic primers for a given (multi-taxon) group of organisms. In both cases, the probe is used to provide quantitative information.

The design of species-specific primers and probes for qPCR comprises the assembly of an inclusive consensus sequence that contains all species variability in a well-known DNA region. For macroorganisms, the mtDNA is preferred since it is more abundant than the nuclear. In addition, there are more sequence data in GenBank. The qPCR primer software application is recommended for the forward and reverse primers and probe generation, which allows for amplification of the target segment of about 100 (90-120) bps. The designed primers and probe with the GenBank database must be double-checked to avoid cross-amplification with other species.

Amplicon sequencing

The amplicons generated by the qPCR are subjected to sequencing. From the First-generation sequencing to the Next-generation sequencing, the DNA sequencing approaches and platforms have undergone tremendous technological development. The history of this methodological approach advancement, presented in Fig. 1.2. covers the:

• First-generation sequencing (FGS) – the shotgun technology approach of Sanger. FGS is the gold sequencing standard that offers high accuracy of long DNA fragments (700 and 1000 bps). However, it is a slow and time-consuming method since only one matrix chain can be detected at a time.
• Second-generation sequencing (SGS)– based on the PCR techniques;
• Third-generation sequencing (TGS) – de novosequencing, based on single-molecule sequencing technology (SMS). The NANOPORE SEQUENCING type of SMS explores electrophoresis to drive individual molecules (one by one) through nanopores for sequencing.
• Next-generation sequencing (NGS) is a massively parallel sequencing technology that offers high speed and scalability of the sequencing process, together with deep sequencing of target DNA regions and a high results preciseness. NGS is less time-consuming and less expensive than the traditional Sanger sequencing approach. NGS uses sequencing by synthesis (SBS, ILLUMINA) and sequencing by ligation (SBL, ROCHE 454) technologies. An SBL modification is the sequencing by oligonucleotide ligation and detection (SOLiD, ABI SOLID), whose detection accuracy is even higher than the other NGS methods. The semiconductor chip technology is based on ion torrent sequencing (ION TORRENT), whose main advantage is its low cost.

Figure. 1.2. DNA sequencing technologies and platforms (Source: Zhang et al., 2021).

Bioinformatic processing of sequenced data, species identification and results interpretation

NGS technology generates big amounts of DNA sequencing data that demand machine-assisted processing and analysis. Here, the in silico approach of bioinformatics helps with sequence sorting, error trimming, and organisms clustering into various taxonomic levels or, in case the taxon isn’t known or the DNA database coverage is poor, into Molecular Operations Taxonomic Units (MOTUs). The bioinformatics approach helps for functional annotation of the obtained sequences that predict genes, operons, and functional RNAs. The protein sequence prediction allows adding members to already-existing groups, creating new orthologous groups, and more precise proteins mapping to distinct orthologous groups. This approach determines whether independent eDNA samples from various environments possess similar functional profiles based on the proteins they encode.

Protocol’s errors-prone steps

• Sampling. The target species determines the time of sampling. It depends on the individual’s history or behavior and must be chosen carefully so as not to compromise the qualitative representation and quantification of the target DNA within the eDNA sample.
• Procedure design. The assay design has to consider both the intra-species and inter-species variations. If a target species’ entire set of genetic variations is not covered, this may lead to false negative results. On the contrary, if the full range of genetic variations in co-occurring and genetically related species is not covered, a false positive result may be registered. That is why it is essential to choose a genetic region that covers the available genetic information for both the target and the related non-target species. Thus, the assay has to be sensitive and specific enough to decrease the jeopardizing results to a minimum.
• Control of the process quality. The assay has to include the proper positive and negative controls for internal verification of results and their quality assurance. These controls have to encompass all process stages:
  • DNA extraction – a negative control to detect cross-contamination among extracts;
  • PCR amplification – internal positive control for each well to signal for reaction inhibition; performance of eDNA extraction and qPCR in a lab where no other DNA samples are handled;
  • Samples of degraded and low-quality DNA should be done in triplicate to avoid false positives;
  • Standard curves should be developed for the DNA obtained from different samples of one target species to extend the range of sample results;
  • Process controls: a negative control comprising species outside the range of the target species and a negative control including samples from distilled water; use of sterile consumables and glassware.
• Detection accuracy. Need of replicate samples to avoid uncertainty and determine the lower limits of detection, which are not known for each species and vary in various factors, such as the species dimensions, density, behavior, environmental habitat.

4.2. Technical challenges and drawbacks

4.2.1. Drawbacks in eDNA obtaining and sequencing

Although those applications of eDNA are indisputable, there are several issues deemed to be problematic and a source of misinterpreted or unreliable results. The main pitfalls, the problems they raise, and the approaches for their resolution are summarized in Table 1.3.

Table 1.3. The main technical pitfalls of eDNA and their resolution approaches.

Pitfall	Problematic issue	Elimination approach
Contamination	– Possible contamination routs: sampling, all laboratory analyses – Cros contamination: mixed DNAs from different localities – Spreading of DNA copies throughout the lab – False positive results	– Minimize the number of sequences obtained in a sample – Perform independent PCR reactions to gain one and the same amplification success – Physically separate the pre-PCR and post-PCR processes – Include blanks for the sampling, DNA extraction, and PCR steps
Enzymes inhibition	– Co-extraction of DNA and humic substances leads to Taq Polymerase inhibition – Generation of false negatives profiles	– Avoid humic acids extraction
PCR and sequencing generated errors	– Point mutations and chimeric molecules formation – False positives during sequencing	– Filter raw sequencing data and practice ‘denoising’ of the signals – Trade-off error trimming and retaining as much sequencing information as possible
Temporal and special consistence of results	– The eDNA degradation time from different habitats varies from several days to centuries resulting in low temporal and special consistence of results – Transport of eDNA within (mainly aquatic) ecosystems	– Careful interpretation of results on spatial and temporal scale
eDNA implementation	– Single-species approach – detects only one species at a time – Multi-species approach (DNA metabarcoding) – amplification of certain sequences (species) less efficiently than others (species) – Reliability of qPCR quantification – definition of true positives from the background – Difficulties in translation of DNA sequence diversity into species diversity due to the limitation of eDNA to short sequences with low taxonomic resolution	– Optimization of generic primers for metabarcoding purposes – Use standardized number of qPCR replicates – generation of model patterns for DNA production and degradation – Filling in the gaps in DNA sequence databases – Standardize the clustering of sequences from metabarcoding data

4.2.2. Data interpretation challenges

The interpretation of eDNA study results demands a robust critical approach. The main challenges for the eDNA data interpretation are related to eDNA detection methods that make no difference between dead and alive organisms, particular life stages (for the animals with sophisticated life cycles), and hybrid species (because of the use of mtDNA that detects only maternal lineage in a hybrid species). On the other hand, eDNA detects just a part of the total number of sites occupied by a given species. In this sense, reliable estimates of species occupancy per eDNA data are needed.

The technical challenges of eDNA data interpretation impose another issue related to standard general protocols elaboration for results authentication of eDNA studies, especially those related to biodiversity representation and conservation.

5. The eDNA applications potential

 5.1. Improvement of eDNA detection methods and protocols

Essential improvement of eDNA methods and protocols to efficiently combat the above-mentioned pitfalls and drawbacks demands future research of eDNA in terrestrial and aquatic ecosystems for biodiversity monitoring to be focused on the following main topics.

• Studying the spatial and temporal distribution of eDNA in various habitats to ensure efficient monitoring of species persistence in space and time;
• Establishment of better-represented links between eDNA quantity and species abundance to allow reliable conclusions on individual density or total biomass presence;
• Precise determination of the eDNA source to minimize the errors in species abundance interpretation;
• Accounting for the impact of the abiotic factors (T ^oC, pH, salinity, UV radiation) influencing eDNA availability and degradation rates;
• Implement technical improvements, such as obtaining longer DNA fragments, targeting nuclear genes, and exploiting eDNA metabarcoding on a wide scale and in more complex ecosystems.

5.2. Advancements in eDNA application as a biodiversity inventory and monitoring tool

eDNA technology offers significant advantages compared to traditional approaches for biodiversity monitoring. Its methodological advancement comprises the following essential characteristics.

• Standardized conditions of the eDNA protocol: from sampling to sequencing data interpretation;
• Non-invasive nature: eDNA technology is absolutely harmless for the species tested and their environmental habitats;
• Independence on seasoning, geographic location, and weather conditions;
• High-resolution of detection. eDNA technology is characterized by great sensitivity. Its methods are proven better than the traditional ones when it is difficult to distinguish and identify species from closely related counterparts.
• Favorable cost-to-value ratio. eDNA offers shorter handling time and lower costs than the traditional biodiversity monitoring approaches. eDNA metabarcoding approach is progressively becoming superior to traditional methods, with the NGS prizes decreasing;
• Emergency of new generations of real-time sequencing technologies and spectroscopic methods.

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