DNA barcoding
DNA barcoding is a method of species identification using a short section of DNA from a specific gene or genes. The premise of DNA barcoding is that by comparison with a reference library of such DNA sections, an individual sequence can be used to uniquely identify an organism to species, just as a supermarket scanner uses the familiar black stripes of the UPC barcode to identify an item in its stock against its reference database. These "barcodes" are sometimes used in an effort to identify unknown species or parts of an organism, simply to catalog as many taxa as possible, or to compare with traditional taxonomy in an effort to determine species boundaries.
Different gene regions are used to identify the different organismal groups using barcoding. The most commonly used barcode region for animals and some protists is a portion of the cytochrome c oxidase I gene, found in mitochondrial DNA. Other genes suitable for DNA barcoding are the internal transcribed spacer rRNA often used for fungi and RuBisCO used for plants. Microorganisms are detected using different gene regions. The 16S rRNA gene for example is widely used in identification of prokaryotes, whereas the 18S rRNA gene is mostly used for detecting microbial eukaryotes. These gene regions are chosen because they have less intraspecific variation than interspecific variation, which is known as the "Barcoding Gap".
Some applications of DNA barcoding include: identifying plant leaves even when flowers or fruits are not available; identifying pollen collected on the bodies of pollinating animals; identifying insect larvae which may have fewer diagnostic characters than adults; or investigating the diet of an animal based on its stomach content, saliva or feces. When barcoding is used to identify organisms from a sample containing DNA from more than one organism, the term DNA metabarcoding is used, e.g. DNA metabarcoding of diatom communities in rivers and streams, which is used to assess water quality.
Background
DNA barcoding techniques were developed from early DNA sequencing work on microbial communities using the 5S rRNA gene. In 2003, specific methods and terminology of modern DNA barcoding were proposed as a standardized method for identifying species, as well as potentially allocating unknown sequences to higher taxa such as orders and phyla, in a paper by Paul D.N. Hebert et al. from the University of Guelph, Ontario, Canada. Hebert and his colleagues demonstrated the utility of the cytochrome c oxidase I gene, first utilized by Folmer et al. in 1994, using their published DNA primers as a tool for phylogenetic analyses at the species levels as a suitable discriminatory tool between metazoan invertebrates. The "Folmer region" of the COI gene is commonly used for distinction between taxa based on its patterns of variation at the DNA level. The relative ease of retrieving the sequence, and variability mixed with conservation between species, are some of the benefits of COI. Calling the profiles "barcodes", Hebert et al. envisaged the development of a COI database that could serve as the basis for a "global bioidentification system".Methods
Sampling and preservation
Barcoding can be done from tissue from a target specimen, from a mixture of organisms, or from DNA present in environmental samples. The methods for sampling, preservation or analysis differ between those different types of sample.Tissue samples
To barcode a tissue sample from the target specimen, a small piece of skin, a scale, a leg or antenna is likely to be sufficient. To avoid contamination, it is necessary to sterilize used tools between samples. It is recommended to collect two samples from one specimen, one to archive, and one for the barcoding process. Sample preservation is crucial to overcome the issue of DNA degradation.
Bulk samples
A bulk sample is a type of environmental sample containing several organisms from the taxonomic group under study. The difference between bulk samples and other environmental samples is that the bulk sample usually provides a large quantity of good-quality DNA. Examples of bulk samples include aquatic macroinvertebrate samples collected by kick-net, or insect samples collected with a Malaise trap. Filtered or size-fractionated water samples containing whole organisms like unicellular eukaryotes are also sometimes defined as bulk samples. Such samples can be collected by the same techniques used to obtain traditional samples for morphology-based identification.
eDNA samples
The environmental DNA method is a non-invasive approach to detect and identify species from cellular debris or extracellular DNA present in environmental samples through barcoding or metabarcoding. The approach is based on the fact that every living organism leaves DNA in the environment, and this environmental DNA can be detected even for organisms that are at very low abundance. Thus, for field sampling, the most crucial part is to use DNA-free material and tools on each sampling site or sample to avoid contamination, if the DNA of the target organism is likely to be present in low quantities. On the other hand, an eDNA sample always includes the DNA of whole-cell, living microorganisms, which are often present in large quantities. Therefore, microorganism samples taken in the natural environment also are called eDNA samples, but contamination is less problematic in this context due to the large quantity of target organisms. The eDNA methods can be used with most sample types, like water, soil, feces, stomach contents, or blood
DNA extraction, amplification and sequencing
DNA barcoding requires that DNA in the sample is extracted. Several different DNA extraction methods exist, and factors like cost, time, sample type and yield affect the selection of the optimal method.When DNA from organismal or eDNA samples is amplified using polymerase chain reaction, the reaction can be affected negatively by inhibitor molecules contained in the sample. Removal of these inhibitors is crucial to ensure that high quality DNA is available for subsequent analyzing.
Amplification of the extracted DNA is a required step in DNA barcoding. Typically, only a small fragment of the total DNA material is sequenced to obtain the DNA barcode and amplification is accordingly limited to the fragment using a pair of primers. Amplification of eDNA material is usually focused on smaller fragment sizes as eDNA is more likely to be fragmented than DNA material from other sources. However, some studies argue that there is no relationship between amplicon size and detection rate of eDNA.
When the DNA barcode marker region has been amplified, the next step is to sequence the marker region using DNA sequencing methods. Many different sequencing platforms are available, and technical development is proceeding rapidly.
Marker selection
Markers used for DNA barcoding are called barcodes. In order to successfully characterize species based on DNA barcodes, selection of informative DNA regions is crucial. A good DNA barcode should have low intra-specific and high inter-specific variability and possess conserved flanking sites for developing universal PCR primers for wide taxonomic application. The goal is to design primers that will detect and distinguish most or all the species in the studied group of organisms. The length of the barcode sequence should be short enough to be used with current sampling source, DNA extraction, amplification and sequencing methods.Ideally, one gene sequence would be used for all taxonomic groups, from viruses to plants and animals. However, no such gene region has been found yet, so different barcodes are used for different groups of organisms, or depending on the study question.
For animals, the most widely used barcode is mitochondrial cytochrome C oxidase I locus. Other mitochondrial genes, such as Cytb, 12S or 16S are also used. Mitochondrial genes are preferred over nuclear genes because of their lack of introns, their haploid mode of inheritance and their limited recombination. Moreover, each cell has various mitochondria and each of them contains several circular DNA molecules. Mitochondria can therefore offer abundant source of DNA even when sample tissue is limited.
In plants, however, mitochondrial genes are not appropriate for DNA barcoding because they exhibit low mutation rates. A few candidate genes have been found in the chloroplast genome, the most promising being maturase K gene by itself or in association with other genes. Multi-locus markers such as ribosomal internal transcribed spacers along with matK, rbcL, trnH or other genes have also been used for species identification. The best discrimination between plant species has been achieved when using two or more chloroplast "barcoding" loci.
For bacteria, the small subunit of ribosomal RNA gene can be used for different taxa, as it is highly conserved. Some studies suggest COI, type II chaperonin or β subunit of RNA polymerase also could serve as bacterial DNA barcodes.
Barcoding fungi is more challenging, and more than one primer combination might be required. The COI marker performs well in certain fungi groups, but not equally well in others. Therefore, additional markers are being used, such as ITS rDNA and the large subunit of nuclear ribosomal RNA.
Within the group of protists, various barcodes have been proposed, such as the D1–D2 or D2–D3 regions of 28S rDNA, V4 subregion of 18S rRNA gene, ITS rDNA and COI. Additionally, some specific barcodes can be used for photosynthetic protists, for example the large subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase gene and the chloroplastic 23S rRNA gene.