Metabarcoding is the barcoding of DNA/RNA (or eDNA/eRNA) in a manner that allows for the simultaneous identification of many taxa within the same sample. The main difference between barcoding and metabarcoding is that metabarcoding does not focus on one specific organism, but instead aims to determine species composition within a sample.
A barcode consists of a short variable gene region (for example, see different markers/barcodes) which is useful for taxonomic assignment flanked by highly conserved gene regions which can be used for primer design. This idea of general barcoding originated in 2003 from researchers at the University of Guelph.
The metabarcoding procedure, like general barcoding, proceeds in order through stages of DNA extraction, PCR amplification, sequencing and data analysis. Different genes are used depending if the aim is to barcode single species or metabarcoding several species. In the latter case, a more universal gene is used. Metabarcoding does not use single species DNA/RNA as a starting point, but DNA/RNA from several different organisms derived from one environmental or bulk sample.
Environmental DNA
Environmental DNA or eDNA describes the genetic material present in environmental samples such as sediment, water, and air, including whole cells, extracellular DNA and potentially whole organisms. eDNA can be captured from environmental samples and preserved, extracted, amplified, sequenced, and categorized based on its sequence. From this information, detection and classification of species is possible. eDNA may come from skin, mucous, saliva, sperm, secretions, eggs, feces, urine, blood, roots, leaves, fruit, pollen, and rotting bodies of larger organisms, while microorganisms may be obtained in their entirety. eDNA production is dependent on biomass, age and feeding activity of the organism as well as physiology, life history, and space use.
By 2019 methods in eDNA research had been expanded to be able to assess whole communities from a single sample. This process involves metabarcoding, which can be precisely defined as the use of general or universal polymerase chain reaction (PCR) primers on mixed DNA samples from any origin followed by high-throughput next-generation sequencing (NGS) to determine the species composition of the sample. This method has been common in microbiology for years, but, as of 2020, it is only just finding its footing in the assessment of macroorganisms. Material was copied from this source, which is available under a Creative Commons Attribution 4.0 International License Ecosystem-wide applications of eDNA metabarcoding have the potential to not only describe communities and biodiversity, but also to detect interactions and functional ecology over large spatial scales, though it may be limited by false readings due to contamination or other errors. Altogether, eDNA metabarcoding increases speed, accuracy, and identification over traditional barcoding and decreases cost, but needs to be standardized and unified, integrating taxonomy and molecular methods for full ecological study.
eDNA metabarcoding has applications to diversity monitoring across all habitats and taxonomic groups, ancient ecosystem reconstruction, plant-pollinator interactions, diet analysis, invasive species detection, pollution responses, and air quality monitoring. eDNA metabarcoding is a unique method still in development and will likely remain in flux for some time as technology advances and procedures become standardized. However, as metabarcoding is optimized and its use becomes more widespread, it is likely to become an essential tool for ecological monitoring and global conservation study.
Community DNA Since the inception of high‐throughput sequencing (HTS), the use of metabarcoding as a biodiversity detection tool has drawn immense interest. However, there has yet to be clarity regarding what source material is used to conduct metabarcoding analyses (e.g., environmental DNA versus community DNA). Without clarity between these two source materials, differences in sampling, as well as differences in laboratory procedures, can impact subsequent bioinformatics pipelines used for data processing, and complicate the interpretation of spatial and temporal biodiversity patterns. Here, we seek to clearly differentiate among the prevailing source materials used and their effect on downstream analysis and interpretation for environmental DNA metabarcoding of animals and plants compared to that of community DNA metabarcoding.
With community DNA metabarcoding of animals and plants, the targeted groups are most often collected in bulk (e.g., soil, malaise trap or net), and individuals are removed from other sample debris and pooled together prior to bulk DNA extraction. In contrast, macro‐organism eDNA is isolated directly from an environmental material (e.g., soil or water) without prior segregation of individual organisms or plant material from the sample and implicitly assumes that the whole organism is not present in the sample. Of course, community DNA samples may contain DNA from parts of tissues, cells and organelles of other organisms (e.g., gut contents, cutaneous intracellular or extracellular DNA). Likewise, macro‐organism eDNA samples may inadvertently capture whole microscopic nontarget organisms (e.g., protists, bacteria). Thus, the distinction can at least partly break down in practice.
Another important distinction between community DNA and macro‐organism eDNA is that sequences generated from community DNA metabarcoding can be taxonomically verified when the specimens are not destroyed in the extraction process. Here, sequences can then be generated from voucher specimens using Sanger sequencing. As the samples for eDNA metabarcoding lack whole organisms, no such in situ comparisons can be made. Taxonomic affinities can therefore only be established by directly comparing obtained sequences (or through bioinformatically generated operational taxonomic units (MOTUs)), to sequences that are taxonomically annotated such as NCBI's GenBank nucleotide database, BOLD, or to self‐generated reference databases from Sanger‐sequenced DNA. (The molecular operational taxonomic unit (MOTU) is a group identified through use of cluster algorithms and a predefined percentage sequence similarity, for example, 97%)). Then, to at least partially corroborate the resulting list of taxa, comparisons are made with conventional physical, acoustic or visual‐based survey methods conducted at the same time or compared with historical records from surveys for a location (see Table 1). A meta-analysis comparing DNA metabarcoding with traditional methods found that the two approaches generally produced similar estimates of taxonomic richness, although they did not always detect the same species. The degree of overlap between the methods varied among organismal groups, with particularly large differences observed for macroinvertebrates and planktonic communities.
The difference in source material between community DNA and eDNA therefore has distinct ramifications for interpreting the scale of inference for time and space about the biodiversity detected. From community DNA, it is clear that the individual species were found in that time and place, but for eDNA, the organism that produced the DNA may be upstream from the sampled location, or the DNA may have been transported in the faeces of a more mobile predatory species (e.g., birds depositing fish eDNA, or was previously present, but no longer active in the community and detection is from DNA that was shed years to decades before. The latter means that the scale of inference both in space and in time must be considered carefully when inferring the presence for the species in the community based on eDNA.
Metabarcoding stages
There are six stages or steps in DNA barcoding and metabarcoding. The DNA barcoding of animals (and specifically of bats) is used as an example in the diagram at the right and in the discussion immediately below.
First, suitable DNA barcoding regions are chosen to answer some specific research question. The most commonly used DNA barcode region for animals is a segment about 600 base pairs long of the mitochondrial gene cytochrome oxidase I (CO1). This locus provides large sequence variation between species yet relatively small amount of variation within species. Other commonly used barcode regions used for species identification of animals are ribosomal DNA (rDNA) regions such as 16S, 18S and 12S and mitochondrial regions such as cytochrome B. These markers have advantages and disadvantages and are used for different purposes. Longer barcode regions (at least 600 base pairs long) are often needed for accurate species delimitation, especially to differentiate close relatives. Identification of the producer of organism's remains such as faeces, hairs and saliva can be used as a proxy measure to verify absence/presence of a species in an ecosystem. The DNA in these remains is usually of low quality and quantity, and therefore, shorter barcodes of around 100 base pairs long are used in these cases. Similarly, DNA remains in dung are often degraded as well, so short barcodes are needed to identify prey consumed.
Second, a reference database needs to be built of all DNA barcodes likely to occur in a study. Ideally, these barcodes need to be generated from vouchered specimens deposited in a publicly accessible place, such as for instance a natural history museum or another research institute. Building up such reference databases is currently being done all over the world. Partner organizations collaborate in international projects such as the International Barcode of Life Project (iBOL) and Consortium for the Barcode of Life (CBOL), aiming to construct a DNA barcode reference that will be the foundation for DNA‐based identification of the world's biome. Well‐known barcode repositories are NCBI GenBank and the Barcode of Life Data System (BOLD).
Third, the cells containing the DNA of interest must be broken open to expose its DNA. This step, DNA extractions and purifications, should be performed from the substrate under investigation. There are several procedures available for this. Specific techniques must be chosen to isolate DNA from substrates with partly degraded DNA, for example fossil samples, and samples containing inhibitors, such as blood, faeces and soil. Extractions in which DNA yield or quality is expected to be low should be carried out in an ancient DNA facility, together with established protocols to avoid contamination with modern DNA. Experiments should always be performed in duplicate and with positive controls included.
Fourth, amplicons have to be generated from DNA extracted, either from a single specimen or from complex mixtures with primers based on DNA barcodes selected under step 1. To keep track of their origin, labelled nucleotides (molecular IDs or MID labels) need to be added in case of metabarcoding. These labels are needed later on in the analyses to trace reads from a bulk data set back to their origin.