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Wikipedia

DNA barcoding

February 16, 2024

Not to be confused with the DNA barcode involved in optical mapping of DNA.

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 (also called "sequences"), 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.[1] 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.[2]

DNA barcoding scheme

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 (COI or COX1) gene, found in mitochondrial DNA. Other genes suitable for DNA barcoding are the internal transcribed spacer (ITS) rRNA often used for fungi and RuBisCO used for plants.[3][4][5] 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 (within species) variation than interspecific (between species) variation, which is known as the "Barcoding Gap".[6]

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.[7] When barcoding is used to identify organisms from a sample containing DNA from more than one organism, the term DNA metabarcoding is used,[8][9] e.g. DNA metabarcoding of diatom communities in rivers and streams, which is used to assess water quality.[10]

Background edit

DNA barcoding techniques were developed from early DNA sequencing work on microbial communities using the 5S rRNA gene.[11] 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.[12] Hebert and his colleagues demonstrated the utility of the cytochrome c oxidase I (COI) gene, first utilized by Folmer et al. in 1994, using their published DNA primers as a tool for phylogenetic analyses at the species levels[12] as a suitable discriminatory tool between metazoan invertebrates.[13] 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 edit

Sampling and preservation edit

Barcoding can be done from tissue from a target specimen, from a mixture of organisms (bulk sample), or from DNA present in environmental samples (e.g. water or soil). 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 (depending on the size of the specimen). 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 (in the sense used here) 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 (eDNA) method is a non-invasive approach to detect and identify species from cellular debris or extracellular DNA present in environmental samples (e.g. water or soil) 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(s) 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 method is applied on most sample types, like water, sediment, soil, animal feces, stomach content or blood from e.g. leeches.[14]

DNA extraction, amplification and sequencing edit

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 (PCR), the reaction can be affected negatively by inhibitor molecules contained in the sample.[15] 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 (typically 400–800 base pairs)[16] to obtain the DNA barcode. Amplification of eDNA material is usually focused on smaller fragment sizes (<200 base pairs), 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.[17][18]

 
HiSeq sequencers at SciLIfeLab in Uppsala, Sweden. The photo was taken during the excursion of SLU course PNS0169 in March 2019.

When the DNA barcode marker region has been amplified, the next step is to sequence the marker region using DNA sequencing methods.[19] Many different sequencing platforms are available, and technical development is proceeding rapidly.

Marker selection edit

 
A schematic view of primers and target region, demonstrated on 16S rRNA gene in Pseudomonas. As primers, one typically selects short conserved sequences with low variability, which can thus amplify most or all species in the chosen target group. The primers are used to amplify a highly variable target region in between the two primers, which is then used for species discrimination. Modified from »Variable Copy Number, Intra-Genomic Heterogeneities and Lateral Transfers of the 16S rRNA Gene in Pseudomonas« by Bodilis, Josselin; Nsigue-Meilo, Sandrine; Besaury, Ludovic; Quillet, Laurent, used under CC BY, available from: https://www.researchgate.net/figure/Hypervariable-regions-within-the-16S-rRNA-gene-in-Pseudomonas-The-plotted-line-reflects_fig2_224832532.

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[12] 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 (high taxonomic resolution). The length of the barcode sequence should be short enough to be used with current sampling source, DNA extraction, amplification and sequencing methods.[20]

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,[citation needed] or depending on the study question.

For animals, the most widely used barcode is mitochondrial cytochrome C oxidase I (COI) locus.[21] 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.[21][22] Moreover, each cell has various mitochondria (up to several thousand) and each of them contains several circular DNA molecules. Mitochondria can therefore offer abundant source of DNA even when sample tissue is limited.[citation needed]

In plants, however, mitochondrial genes are not appropriate for DNA barcoding because they exhibit low mutation rates.[23] A few candidate genes have been found in the chloroplast genome, the most promising being maturase K gene (matK) by itself or in association with other genes. Multi-locus markers such as ribosomal internal transcribed spacers (ITS DNA) along with matK, rbcL, trnH or other genes have also been used for species identification.[citation needed] The best discrimination between plant species has been achieved when using two or more chloroplast barcodes.[24]

For bacteria, the small subunit of ribosomal RNA (16S) gene can be used for different taxa, as it is highly conserved.[25] Some studies suggest COI,[26] type II chaperonin (cpn60)[27] or β subunit of RNA polymerase (rpoB)[28] also could serve as bacterial DNA barcodes.

Barcoding fungi is more challenging, and more than one primer combination might be required.[29] The COI marker performs well in certain fungi groups,[30] but not equally well in others.[31] Therefore, additional markers are being used, such as ITS rDNA and the large subunit of nuclear ribosomal RNA (28S LSU rRNA).[32]

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 (rbcL) and the chloroplastic 23S rRNA gene.[citation needed]

Reference libraries and bioinformatics edit

Reference libraries are used for the taxonomic identification, also called annotation, of sequences obtained from barcoding or metabarcoding. These databases contain the DNA barcodes assigned to previously identified taxa. Most reference libraries do not cover all species within an organism group, and new entries are continually created. In the case of macro- and many microorganisms (such as algae), these reference libraries require detailed documentation (sampling location and date, person who collected it, image, etc.) and authoritative taxonomic identification of the voucher specimen, as well as submission of sequences in a particular format. However, such standards are fulfilled for only a small number of species. The process also requires the storage of voucher specimens in museum collections, herbaria and other collaborating institutions. Both taxonomically comprehensive coverage and content quality are important for identification accuracy.[33] In the microbial world, there is no DNA information for most species names, and many DNA sequences cannot be assigned to any Linnaean binomial.[34] Several reference databases exist depending on the organism group and the genetic marker used. There are smaller, national databases (e.g. FinBOL), and large consortia like the International Barcode of Life Project (iBOL).[35]

BOLD

Launched in 2007, the Barcode of Life Data System (BOLD)[36] is one of the biggest databases, containing about 780 000 BINs (Barcode Index Numbers) in 2022. It is a freely accessible repository for the specimen and sequence records for barcode studies, and it is also a workbench aiding the management, quality assurance and analysis of barcode data. The database mainly contains BIN records for animals based on the COI genetic marker. For plant identification, BOLD accepts sequences from matK and rbcL.

UNITE

The UNITE database[37] was launched in 2003 and is a reference database for the molecular identification of fungal (and since 2018 all eukaryotic) species with the nuclear ribosomal internal transcribed spacer (ITS) genetic marker region. This database is based on the concept of species hypotheses: you choose the % at which you want to work, and the sequences are sorted in comparison to sequences obtained from voucher specimens identified by experts.

Diat.barcode[permanent dead link]

Diat.barcode[38] database was first published under the name R-syst::diatom[39] in 2016 starting with data from two sources: the Thonon culture collection (TCC) in the hydrobiological station of the French National Institute for Agricultural Research (INRA), and from the NCBI (National Center for Biotechnology Information) nucleotide database. Diat.barcode provides data for two genetic markers, rbcL (Ribulose-1,5-bisphosphate carboxylase/oxygenase) and 18S (18S ribosomal RNA). The database also involves additional, trait information of species, like morphological characteristics (biovolume, size dimensions, etc.), life-forms (mobility, colony-type, etc.) or ecological features (pollution sensitivity, etc.).

Bioinformatic analysis edit

In order to obtain well structured, clean and interpretable data, raw sequencing data must be processed using bioinformatic analysis. The FASTQ file with the sequencing data contains two types of information: the sequences detected in the sample (FASTA file) and a quality file with quality scores (PHRED scores) associated with each nucleotide of each DNA sequence. The PHRED scores indicate the probability with which the associated nucleotide has been correctly scored.

PHRED quality score and the associated certainty level
10 90%
20 99%
30 99.9%
40 99.99%
50 99.999%

In general, the PHRED score decreases towards the end of each DNA sequence. Thus some bioinformatics pipelines simply cut the end of the sequences at a defined threshold.

Some sequencing technologies, like MiSeq, use paired-end sequencing during which sequencing is performed from both directions producing better quality. The overlapping sequences are then aligned into contigs and merged. Usually, several samples are pooled in one run, and each sample is characterized by a short DNA fragment, the tag. In a demultiplexing step, sequences are sorted using these tags to reassemble the separate samples. Before further analysis, tags and other adapters are removed from the barcoding sequence DNA fragment. During trimming, the bad quality sequences (low PHRED scores), or sequences that are much shorter or longer than the targeted DNA barcode, are removed. The following dereplication step is the process where all of the quality-filtered sequences are collapsed into a set of unique reads (individual sequence units ISUs) with the information of their abundance in the samples. After that, chimeras (i.e. compound sequences formed from pieces of mixed origin) are detected and removed. Finally, the sequences are clustered into OTUs (Operational Taxonomic Units), using one of many clustering strategies. The most frequently used bioinformatic software include Mothur,[40] Uparse,[41] Qiime,[42] Galaxy,[43] Obitools,[44] JAMP,[45] Barque,[46] and DADA2.[47]

Comparing the abundance of reads, i.e. sequences, between different samples is still a challenge because both the total number of reads in a sample as well as the relative amount of reads for a species can vary between samples, methods, or other variables. For comparison, one may then reduce the number of reads of each sample to the minimal number of reads of the samples to be compared – a process called rarefaction. Another way is to use the relative abundance of reads.[48]

Species identification and taxonomic assignment edit

The taxonomic assignment of the OTUs to species is achieved by matching of sequences to reference libraries. The Basic Local Alignment Search Tool (BLAST) is commonly used to identify regions of similarity between sequences by comparing sequence reads from the sample to sequences in reference databases.[49] If the reference database contains sequences of the relevant species, then the sample sequences can be identified to species level. If a sequence cannot be matched to an existing reference library entry, DNA barcoding can be used to create a new entry.

In some cases, due to the incompleteness of reference databases, identification can only be achieved at higher taxonomic levels, such as assignment to a family or class. In some organism groups such as bacteria, taxonomic assignment to species level is often not possible. In such cases, a sample may be assigned to a particular operational taxonomic unit (OTU).

Applications edit

Applications of DNA barcoding include identification of new species, safety assessment of food, identification and assessment of cryptic species, detection of alien species, identification of endangered and threatened species,[50] linking egg and larval stages to adult species, securing intellectual property rights for bioresources, framing global management plans for conservation strategies, elucidate feeding niches,[51] and forensic science.[52] DNA barcode markers can be applied to address basic questions in systematics, ecology, evolutionary biology and conservation, including community assembly, species interaction networks, taxonomic discovery, and assessing priority areas for environmental protection.

Identification of species edit

Specific short DNA sequences or markers from a standardized region of the genome can provide a DNA barcode for identifying species.[53] Molecular methods are especially useful when traditional methods are not applicable. DNA barcoding has great applicability in identification of larvae for which there are generally few diagnostic characters available, and in association of different life stages (e.g. larval and adult) in many animals.[54] Identification of species listed in the Convention of the International Trade of Endangered Species (CITES) appendixes using barcoding techniques is used in monitoring of illegal trade.[55]

Detection of invasive species edit

Alien species can be detected via barcoding.[56][57] Barcoding can be suitable for detection of species in e.g. border control, where rapid and accurate morphological identification is often not possible due to similarities between different species, lack of sufficient diagnostic characteristics[56] and/or lack of taxonomic expertise. Barcoding and metabarcoding can also be used to screen ecosystems for invasive species, and to distinguish between an invasive species and native, morphologically similar, species.[58] The high efficiency of DNA identification is shown relative to the traditional monitoring of biological invasions.[59]

Delimiting cryptic species edit

DNA barcoding enables the identification and recognition of cryptic species.[60] The results of DNA barcoding analyses depend however upon the choice of analytical methods, so the process of delimiting cryptic species using DNA barcodes can be as subjective as any other form of taxonomy. Hebert et al. (2004) concluded that the butterfly Astraptes fulgerator in north-western Costa Rica actually consists of 10 different species.[61] These results, however, were subsequently challenged by Brower (2006), who pointed out numerous serious flaws in the analysis, and concluded that the original data could support no more than the possibility of three to seven cryptic taxa rather than ten cryptic species.[62] Smith et al. (2007) used cytochrome c oxidase I DNA barcodes for species identification of the 20 morphospecies of Belvosia parasitoid flies (Diptera: Tachinidae) reared from caterpillars (Lepidoptera) in Area de Conservación Guanacaste (ACG), northwestern Costa Rica. These authors discovered that barcoding raises the species count to 32, by revealing that each of the three parasitoid species, previously considered as generalists, actually are arrays of highly host-specific cryptic species.[63] For 15 morphospecies of polychaetes within the deep Antarctic benthos studied through DNA barcoding, cryptic diversity was found in 50% of the cases. Furthermore, 10 previously overlooked morphospecies were detected, increasing the total species richness in the sample by 233%.[64]

 
Barcoding is a tool to vouch for food quality. Here, DNA from traditional Norwegian Christmas food is extracted at the molecular systematic lab at NTNU University Museum.

Diet analysis and food web application edit

DNA barcoding and metabarcoding can be useful in diet analysis studies,[65] and is typically used if prey specimens cannot be identified based on morphological characters.[66][67] There is a range of sampling approaches in diet analysis: DNA metabarcoding can be conducted on stomach contents,[68] feces,[67][69] saliva[70] or whole body analysis.[50][71] In fecal samples or highly digested stomach contents, it is often not possible to distinguish tissue from single species, and therefore metabarcoding can be applied instead.[67][72] Feces or saliva represent non-invasive sampling approaches, while whole body analysis often means that the individual needs to be killed first. For smaller organisms, sequencing for stomach content is then often done by sequencing the entire animal.

Barcoding for food safety edit

DNA barcoding represents an essential tool to evaluate the quality of food products. The purpose is to guarantee food traceability, to minimize food piracy, and to valuate local and typical agro-food production. Another purpose is to safeguard public health; for example, metabarcoding offers the possibility to identify groupers causing Ciguatera fish poisoning from meal remnants,[73] or to separate poisonous mushrooms from edible ones (Ref).

Biomonitoring and ecological assessment edit

DNA barcoding can be used to assess the presence of endangered species for conservation efforts (Ref), or the presence of indicator species reflective to specific ecological conditions (Ref), for example excess nutrients or low oxygen levels.

Forensic Science edit

DNA barcoding is often used for species identification in forensic science cases. Unknown animal or plant samples at crime scenes can be found, collected, and identified, in hopes of linking it to a suspect and getting a conviction.[74] Poaching, killing of endangered species, and animal abuse are examples of crimes where DNA barcoding is used, since animal DNA is often found.[52][75] On the other hand, plant DNA is usually used as trace evidence to link a suspect to a crime scene.[76]

Potentials and shortcomings edit

Potentials edit

Traditional bioassessment methods are well established internationally, and serve biomonitoring well, as for example for aquatic bioassessment within the EU Directives WFD and MSFD. However, DNA barcoding could improve traditional methods for the following reasons; DNA barcoding (i) can increase taxonomic resolution and harmonize the identification of taxa which are difficult to identify or lack experts, (ii) can more accurately/precisely relate environmental factors to specific taxa (iii) can increase comparability among regions, (iv) allows for the inclusion of early life stages and fragmented specimens, (v) allows delimitation of cryptic/rare species (vi) allows for development of new indices e.g. rare/cryptic species which may be sensitive/tolerant to stressors, (vii) increases the number of samples which can be processed and reduces processing time resulting in increased knowledge of species ecology, (viii) is a non-invasive way of monitoring when using eDNA methods.[77]

Time and cost edit

DNA barcoding is faster than traditional morphological methods all the way from training through to taxonomic assignment. It takes less time to gain expertise in DNA methods than becoming an expert in taxonomy. In addition, the DNA barcoding workflow (i.e. from sample to result) is generally quicker than traditional morphological workflow and allows the processing of more samples.

Taxonomic resolution edit

DNA barcoding allows the resolution of taxa from higher (e.g. family) to lower (e.g. species) taxonomic levels, that are otherwise too difficult to identify using traditional morphological methods, like e.g. identification via microscopy. For example, Chironomidae (the non-biting midge) are widely distributed in both terrestrial and freshwater ecosystems. Their richness and abundance make them important for ecological processes and networks, and they are one of many invertebrate groups used in biomonitoring. Invertebrate samples can contain as many as 100 species of chironomids which often make up as much as 50% of a sample. Despite this, they are usually not identified below the family level because of the taxonomic expertise and time required.[78] This may result in different chironomid species with different ecological preferences grouped together, resulting in inaccurate assessment of water quality.

DNA barcoding provides the opportunity to resolve taxa, and directly relate stressor effects to specific taxa such as individual chironomid species. For example, Beermann et al. (2018) DNA barcoded Chironomidae to investigate their response to multiple stressors; reduced flow, increased fine-sediment and increased salinity.[79] After barcoding, it was found that the chironomid sample consisted of 183 Operational Taxonomic Units (OTUs), i.e. barcodes (sequences) that are often equivalent to morphological species. These 183 OTUs displayed 15 response types rather than the previously reported [80] two response types recorded when all chironomids were grouped together in the same multiple stressor study. A similar trend was discovered in a study by Macher et al. (2016) which discovered cryptic diversity within the New Zealand mayfly species Deleatidium sp. This study found different response patterns of 12 molecular distinct OTUs to stressors which may change the consensus that this mayfly is sensitive to pollution.[81]

Shortcomings edit

Despite the advantages offered by DNA barcoding, it has also been suggested that DNA barcoding is best used as a complement to traditional morphological methods.[77] This recommendation is based on multiple perceived challenges.

Physical parameters edit

It is not completely straightforward to connect DNA barcodes with ecological preferences of the barcoded taxon in question, as is needed if barcoding is to be used for biomonitoring. For example, detecting target DNA in aquatic systems depends on the concentration of DNA molecules at a site, which in turn can be affected by many factors. The presence of DNA molecules also depends on dispersion at a site, e.g. direction or strength of currents. It is not really known how DNA moves around in streams and lakes, which makes sampling difficult. Another factor might be the behavior of the target species, e.g. fish can have seasonal changes of movements, crayfish or mussels will release DNA in larger amounts just at certain times of their life (moulting, spawning). For DNA in soil, even less is known about distribution, quantity or quality.

The major limitation of the barcoding method is that it relies on barcode reference libraries for the taxonomic identification of the sequences. The taxonomic identification is accurate only if a reliable reference is available. However, most databases are still incomplete, especially for smaller organisms e.g. fungi, phytoplankton, nematoda etc. In addition, current databases contain misidentifications, spelling mistakes and other errors. There is massive curation and completion effort around the databases for all organisms necessary, involving large barcoding projects (for example the iBOL project for the Barcode of Life Data Systems (BOLD) reference database).[82][83] However, completion and curation are difficult and time-consuming. Without vouchered specimens, there can be no certainty about whether the sequence used as a reference is correct.

DNA sequence databases like GenBank contain many sequences that are not tied to vouchered specimens (for example, herbarium specimens, cultured cell lines, or sometimes images). This is problematic in the face of taxonomic issues such as whether several species should be split or combined, or whether past identifications were sound. Reusing sequences, not tied to vouchered specimens, of initially misidentified organism may support incorrect conclusions and must be avoided.[84] Therefore, best practice for DNA barcoding is to sequence vouchered specimens.[85][86] For many taxa, it can be however difficult to obtain reference specimens, for example with specimens that are difficult to catch, available specimens are poorly conserved, or adequate taxonomic expertise is lacking.[84]

Importantly, DNA barcodes can also be used to create interim taxonomy, in which case OTUs can be used as substitutes for traditional Latin binomials – thus significantly reducing dependency on fully populated reference databases.[87]

Technological bias edit

DNA barcoding also carries methodological bias, from sampling to bioinformatics data analysis. Beside the risk of contamination of the DNA sample by PCR inhibitors, primer bias is one of the major sources of errors in DNA barcoding.[88][89] The isolation of an efficient DNA marker and the design of primers is a complex process and considerable effort has been made to develop primers for DNA barcoding in different taxonomic groups.[90] However, primers will often bind preferentially to some sequences, leading to differential primer efficiency and specificity and unrepresentative communities’ assessment and richness inflation.[91] Thus, the composition of the sample's communities sequences is mainly altered at the PCR step.  Besides, PCR replication is often required, but leads to an exponential increase in the risk of contamination. Several studies have highlighted the possibility to use mitochondria-enriched samples [92][93] or PCR-free approaches to avoid these biases, but as of today, the DNA metabarcoding technique is still based on the sequencing of amplicons.[90] Other bias enter the picture during the sequencing and during the bioinformatic processing of the sequences, like the creation of chimeras.

Lack of standardization edit

Even as DNA barcoding is more widely used and applied, there is no agreement concerning the methods for DNA preservation or extraction, the choices of DNA markers and primers set, or PCR protocols. The parameters of bioinformatics pipelines (for example OTU clustering, taxonomic assignment algorithms or thresholds etc.) are at the origin of much debate among DNA barcoding users.[90] Sequencing technologies are also rapidly evolving, together with the tools for the analysis of the massive amounts of DNA data generated, and standardization of the methods is urgently needed to enable collaboration and data sharing at greater spatial and time-scale. This standardisation of barcoding methods at the European scale is part of the objectives of the European COST Action DNAqua-net [94] and is also addressed by CEN (the European Committee for Standardization).[95]

Another criticism of DNA barcoding is its limited efficiency for accurate discrimination below species level (for example, to distinguish between varieties), for hybrid detection, and that it can be affected by evolutionary rates[citation needed].

Mismatches between conventional (morphological) and barcode based identification edit

It is important to know that taxa lists derived by conventional (morphological) identification are not, and maybe never will be, directly comparable to taxa lists derived from barcode based identification because of several reasons. The most important cause is probably the incompleteness and lack of accuracy of the molecular reference databases preventing a correct taxonomic assignment of eDNA sequences. Taxa not present in reference databases will not be found by eDNA, and sequences linked to a wrong name will lead to incorrect identification.[77] Other known causes are a different sampling scale and size between a traditional and a molecular sample, the possible analysis of dead organisms, which can happen in different ways for both methods depending on organism group, and the specific selection of identification in either method, i.e. varying taxonomical expertise or possibility to identify certain organism groups, respectively primer bias leading also to a potential biased analysis of taxa.[77]

Estimates of richness/diversity edit

DNA Barcoding can result in an over or underestimate of species richness and diversity. Some studies suggest that artifacts (identification of species not present in a community) are a major cause of inflated biodiversity.[96][97] The most problematic issue are taxa represented by low numbers of sequencing reads. These reads are usually removed during the data filtering process, since different studies suggest that most of these low-frequency reads may be artifacts.[98] However, real rare taxa may exist among these low-abundance reads.[99] Rare sequences can reflect unique lineages in communities which make them informative and valuable sequences. Thus, there is a strong need for more robust bioinformatics algorithms that allow the differentiation between informative reads and artifacts. Complete reference libraries would also allow a better testing of bioinformatics algorithms, by permitting a better filtering of artifacts (i.e. the removal of sequences lacking a counterpart among extant species) and therefore, it would be possible obtain a more accurate species assignment.[100] Cryptic diversity can also result in inflated biodiversity as one morphological species may actually split into many distinct molecular sequences.[77] This will go a long way in generating DNA reference data which is crucial for environmental DNA-based biodiversity monitoring.

Megabarcoding edit

Megabarcoding is a term used to describe high-throughput specimen-based DNA barcoding, where thousands of specimens can be barcoded simultaneously for species identification and discovery.[101][102][103][104][105]

 
Megabarcoding workflow

This is enabled by the use of third-generation sequencing platforms including PacBio (Sequel I/II) by Pacific Biosciences and MinION, PromethION by Oxford Nanopore Technology. As compared to Sanger sequencing, megabarcoding is faster and cheaper, allowing for the large-scale generation of DNA barcodes for thousands of species.[106]

Applications edit

Megabarcoding can help fill the dark taxa. DNA barcode reference data gap for insects and accelerate species discovery,[107][108] understand species diversity patterns,[109][110][111] evaluate species richness,[112] generate rapid biodiversity species inventories,[113] track baseline shifts,[114] and matching life-history stages.[115]

Metabarcoding edit

 
Differences in the standard methods for DNA barcoding and metabarcoding. While DNA barcoding points to find a specific species, metabarcoding looks for the whole community.
Main article: Metabarcoding

Metabarcoding is defined as the barcoding of DNA or eDNA (environmental DNA) that allows for simultaneous identification of many taxa within the same (environmental) sample, however often within the same organism group. The main difference between the approaches is that metabarcoding, in contrast to barcoding, does not focus on one specific organism, but instead aims to determine species composition within a sample.

Methodology edit

The metabarcoding procedure, like general barcoding, covers the steps of DNA extraction, PCR amplification, sequencing and data analysis. 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.[116] 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.

Applications edit

Metabarcoding has the potential to complement biodiversity measures, and even replace them in some instances, especially as the technology advances and procedures gradually become cheaper, more optimized and widespread.[117][118]

DNA metabarcoding applications include Biodiversity monitoring in terrestrial and aquatic environments, Paleontology and ancient ecosystems, Plant-pollinator interactions, Diet analysis and Food safety.

Advantages and challenges edit

The general advantages and shortcomings for barcoding reviewed above are valid also for metabarcoding. One particular drawback for metabarcoding studies is that there is no consensus yet regarding the optimal experimental design and bioinformatics criteria to be applied in eDNA metabarcoding.[119] However, there are current joined attempts, like e.g. the EU COST network DNAqua-Net, to move forward by exchanging experience and knowledge to establish best-practice standards for biomonitoring.[77]

Artificial DNA barcoding edit

In 2014, researchers from ETH Zurich suggested using artificial, sub-micrometer-sized DNA barcodes as an "invisible oil tag". The barcodes consist of synthetic DNA sequences inside magnetically recoverable silica particles. They can be added to food oil in a very small amount (down to 1 ppb) as a label, and can be retrieved at any time for authenticity test by PCR/sequencing. This method can be used to test olive oil for adulteration.[120]

See also edit

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References edit

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External links edit

  • SweBOL
  • FinBOL
  • International Barcode of Life Project (iBOL)
  • BOLD
  • UNITE
  • Diat.barcode[permanent dead link]

February 16, 2024
barcoding, confused, with, barcode, involved, optical, mapping, method, species, identification, using, short, section, from, specific, gene, genes, premise, that, comparison, with, reference, library, such, sections, also, called, sequences, individual, seque. Not to be confused with the DNA barcode involved in optical mapping of DNA 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 also called sequences 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 1 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 2 DNA barcoding schemeDifferent 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 COI or COX1 gene found in mitochondrial DNA Other genes suitable for DNA barcoding are the internal transcribed spacer ITS rRNA often used for fungi and RuBisCO used for plants 3 4 5 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 within species variation than interspecific between species variation which is known as the Barcoding Gap 6 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 7 When barcoding is used to identify organisms from a sample containing DNA from more than one organism the term DNA metabarcoding is used 8 9 e g DNA metabarcoding of diatom communities in rivers and streams which is used to assess water quality 10 Contents 1 Background 2 Methods 2 1 Sampling and preservation 2 2 DNA extraction amplification and sequencing 2 3 Marker selection 3 Reference libraries and bioinformatics 3 1 Bioinformatic analysis 3 2 Species identification and taxonomic assignment 4 Applications 4 1 Identification of species 4 2 Detection of invasive species 4 3 Delimiting cryptic species 4 4 Diet analysis and food web application 4 5 Barcoding for food safety 4 6 Biomonitoring and ecological assessment 4 7 Forensic Science 5 Potentials and shortcomings 5 1 Potentials 5 1 1 Time and cost 5 1 2 Taxonomic resolution 5 2 Shortcomings 5 2 1 Physical parameters 5 2 2 Technological bias 5 2 3 Lack of standardization 5 2 4 Mismatches between conventional morphological and barcode based identification 5 2 5 Estimates of richness diversity 6 Megabarcoding 6 1 Applications 7 Metabarcoding 7 1 Methodology 7 2 Applications 7 3 Advantages and challenges 8 Artificial DNA barcoding 9 See also 10 References 11 External linksBackground editDNA barcoding techniques were developed from early DNA sequencing work on microbial communities using the 5S rRNA gene 11 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 12 Hebert and his colleagues demonstrated the utility of the cytochrome c oxidase I COI gene first utilized by Folmer et al in 1994 using their published DNA primers as a tool for phylogenetic analyses at the species levels 12 as a suitable discriminatory tool between metazoan invertebrates 13 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 editSampling and preservation edit Barcoding can be done from tissue from a target specimen from a mixture of organisms bulk sample or from DNA present in environmental samples e g water or soil The methods for sampling preservation or analysis differ between those different types of sample Tissue samplesTo barcode a tissue sample from the target specimen a small piece of skin a scale a leg or antenna is likely to be sufficient depending on the size of the specimen 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 samplesA bulk sample is a type of environmental sample containing several organisms from the taxonomic group under study The difference between bulk samples in the sense used here 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 samplesThe environmental DNA eDNA method is a non invasive approach to detect and identify species from cellular debris or extracellular DNA present in environmental samples e g water or soil 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 s 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 method is applied on most sample types like water sediment soil animal feces stomach content or blood from e g leeches 14 DNA extraction amplification and sequencing edit 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 PCR the reaction can be affected negatively by inhibitor molecules contained in the sample 15 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 typically 400 800 base pairs 16 to obtain the DNA barcode Amplification of eDNA material is usually focused on smaller fragment sizes lt 200 base pairs 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 17 18 nbsp HiSeq sequencers at SciLIfeLab in Uppsala Sweden The photo was taken during the excursion of SLU course PNS0169 in March 2019 When the DNA barcode marker region has been amplified the next step is to sequence the marker region using DNA sequencing methods 19 Many different sequencing platforms are available and technical development is proceeding rapidly Marker selection edit nbsp A schematic view of primers and target region demonstrated on 16S rRNA gene in Pseudomonas As primers one typically selects short conserved sequences with low variability which can thus amplify most or all species in the chosen target group The primers are used to amplify a highly variable target region in between the two primers which is then used for species discrimination Modified from Variable Copy Number Intra Genomic Heterogeneities and Lateral Transfers of the 16S rRNA Gene in Pseudomonas by Bodilis Josselin Nsigue Meilo Sandrine Besaury Ludovic Quillet Laurent used under CC BY available from https www researchgate net figure Hypervariable regions within the 16S rRNA gene in Pseudomonas The plotted line reflects fig2 224832532 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 12 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 high taxonomic resolution The length of the barcode sequence should be short enough to be used with current sampling source DNA extraction amplification and sequencing methods 20 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 citation needed or depending on the study question For animals the most widely used barcode is mitochondrial cytochrome C oxidase I COI locus 21 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 21 22 Moreover each cell has various mitochondria up to several thousand and each of them contains several circular DNA molecules Mitochondria can therefore offer abundant source of DNA even when sample tissue is limited citation needed In plants however mitochondrial genes are not appropriate for DNA barcoding because they exhibit low mutation rates 23 A few candidate genes have been found in the chloroplast genome the most promising being maturase K gene matK by itself or in association with other genes Multi locus markers such as ribosomal internal transcribed spacers ITS DNA along with matK rbcL trnH or other genes have also been used for species identification citation needed The best discrimination between plant species has been achieved when using two or more chloroplast barcodes 24 For bacteria the small subunit of ribosomal RNA 16S gene can be used for different taxa as it is highly conserved 25 Some studies suggest COI 26 type II chaperonin cpn60 27 or b subunit of RNA polymerase rpoB 28 also could serve as bacterial DNA barcodes Barcoding fungi is more challenging and more than one primer combination might be required 29 The COI marker performs well in certain fungi groups 30 but not equally well in others 31 Therefore additional markers are being used such as ITS rDNA and the large subunit of nuclear ribosomal RNA 28S LSU rRNA 32 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 rbcL and the chloroplastic 23S rRNA gene citation needed Reference libraries and bioinformatics editReference libraries are used for the taxonomic identification also called annotation of sequences obtained from barcoding or metabarcoding These databases contain the DNA barcodes assigned to previously identified taxa Most reference libraries do not cover all species within an organism group and new entries are continually created In the case of macro and many microorganisms such as algae these reference libraries require detailed documentation sampling location and date person who collected it image etc and authoritative taxonomic identification of the voucher specimen as well as submission of sequences in a particular format However such standards are fulfilled for only a small number of species The process also requires the storage of voucher specimens in museum collections herbaria and other collaborating institutions Both taxonomically comprehensive coverage and content quality are important for identification accuracy 33 In the microbial world there is no DNA information for most species names and many DNA sequences cannot be assigned to any Linnaean binomial 34 Several reference databases exist depending on the organism group and the genetic marker used There are smaller national databases e g FinBOL and large consortia like the International Barcode of Life Project iBOL 35 BOLDLaunched in 2007 the Barcode of Life Data System BOLD 36 is one of the biggest databases containing about 780 000 BINs Barcode Index Numbers in 2022 It is a freely accessible repository for the specimen and sequence records for barcode studies and it is also a workbench aiding the management quality assurance and analysis of barcode data The database mainly contains BIN records for animals based on the COI genetic marker For plant identification BOLD accepts sequences from matK and rbcL UNITEThe UNITE database 37 was launched in 2003 and is a reference database for the molecular identification of fungal and since 2018 all eukaryotic species with the nuclear ribosomal internal transcribed spacer ITS genetic marker region This database is based on the concept of species hypotheses you choose the at which you want to work and the sequences are sorted in comparison to sequences obtained from voucher specimens identified by experts Diat barcode permanent dead link Diat barcode 38 database was first published under the name R syst diatom 39 in 2016 starting with data from two sources the Thonon culture collection TCC in the hydrobiological station of the French National Institute for Agricultural Research INRA and from the NCBI National Center for Biotechnology Information nucleotide database Diat barcode provides data for two genetic markers rbcL Ribulose 1 5 bisphosphate carboxylase oxygenase and 18S 18S ribosomal RNA The database also involves additional trait information of species like morphological characteristics biovolume size dimensions etc life forms mobility colony type etc or ecological features pollution sensitivity etc Bioinformatic analysis edit In order to obtain well structured clean and interpretable data raw sequencing data must be processed using bioinformatic analysis The FASTQ file with the sequencing data contains two types of information the sequences detected in the sample FASTA file and a quality file with quality scores PHRED scores associated with each nucleotide of each DNA sequence The PHRED scores indicate the probability with which the associated nucleotide has been correctly scored PHRED quality score and the associated certainty level 10 90 20 99 30 99 9 40 99 99 50 99 999 In general the PHRED score decreases towards the end of each DNA sequence Thus some bioinformatics pipelines simply cut the end of the sequences at a defined threshold Some sequencing technologies like MiSeq use paired end sequencing during which sequencing is performed from both directions producing better quality The overlapping sequences are then aligned into contigs and merged Usually several samples are pooled in one run and each sample is characterized by a short DNA fragment the tag In a demultiplexing step sequences are sorted using these tags to reassemble the separate samples Before further analysis tags and other adapters are removed from the barcoding sequence DNA fragment During trimming the bad quality sequences low PHRED scores or sequences that are much shorter or longer than the targeted DNA barcode are removed The following dereplication step is the process where all of the quality filtered sequences are collapsed into a set of unique reads individual sequence units ISUs with the information of their abundance in the samples After that chimeras i e compound sequences formed from pieces of mixed origin are detected and removed Finally the sequences are clustered into OTUs Operational Taxonomic Units using one of many clustering strategies The most frequently used bioinformatic software include Mothur 40 Uparse 41 Qiime 42 Galaxy 43 Obitools 44 JAMP 45 Barque 46 and DADA2 47 Comparing the abundance of reads i e sequences between different samples is still a challenge because both the total number of reads in a sample as well as the relative amount of reads for a species can vary between samples methods or other variables For comparison one may then reduce the number of reads of each sample to the minimal number of reads of the samples to be compared a process called rarefaction Another way is to use the relative abundance of reads 48 Species identification and taxonomic assignment edit The taxonomic assignment of the OTUs to species is achieved by matching of sequences to reference libraries The Basic Local Alignment Search Tool BLAST is commonly used to identify regions of similarity between sequences by comparing sequence reads from the sample to sequences in reference databases 49 If the reference database contains sequences of the relevant species then the sample sequences can be identified to species level If a sequence cannot be matched to an existing reference library entry DNA barcoding can be used to create a new entry In some cases due to the incompleteness of reference databases identification can only be achieved at higher taxonomic levels such as assignment to a family or class In some organism groups such as bacteria taxonomic assignment to species level is often not possible In such cases a sample may be assigned to a particular operational taxonomic unit OTU Applications editApplications of DNA barcoding include identification of new species safety assessment of food identification and assessment of cryptic species detection of alien species identification of endangered and threatened species 50 linking egg and larval stages to adult species securing intellectual property rights for bioresources framing global management plans for conservation strategies elucidate feeding niches 51 and forensic science 52 DNA barcode markers can be applied to address basic questions in systematics ecology evolutionary biology and conservation including community assembly species interaction networks taxonomic discovery and assessing priority areas for environmental protection Identification of species edit Specific short DNA sequences or markers from a standardized region of the genome can provide a DNA barcode for identifying species 53 Molecular methods are especially useful when traditional methods are not applicable DNA barcoding has great applicability in identification of larvae for which there are generally few diagnostic characters available and in association of different life stages e g larval and adult in many animals 54 Identification of species listed in the Convention of the International Trade of Endangered Species CITES appendixes using barcoding techniques is used in monitoring of illegal trade 55 Detection of invasive species edit Alien species can be detected via barcoding 56 57 Barcoding can be suitable for detection of species in e g border control where rapid and accurate morphological identification is often not possible due to similarities between different species lack of sufficient diagnostic characteristics 56 and or lack of taxonomic expertise Barcoding and metabarcoding can also be used to screen ecosystems for invasive species and to distinguish between an invasive species and native morphologically similar species 58 The high efficiency of DNA identification is shown relative to the traditional monitoring of biological invasions 59 Delimiting cryptic species edit DNA barcoding enables the identification and recognition of cryptic species 60 The results of DNA barcoding analyses depend however upon the choice of analytical methods so the process of delimiting cryptic species using DNA barcodes can be as subjective as any other form of taxonomy Hebert et al 2004 concluded that the butterfly Astraptes fulgerator in north western Costa Rica actually consists of 10 different species 61 These results however were subsequently challenged by Brower 2006 who pointed out numerous serious flaws in the analysis and concluded that the original data could support no more than the possibility of three to seven cryptic taxa rather than ten cryptic species 62 Smith et al 2007 used cytochrome c oxidase I DNA barcodes for species identification of the 20 morphospecies of Belvosia parasitoid flies Diptera Tachinidae reared from caterpillars Lepidoptera in Area de Conservacion Guanacaste ACG northwestern Costa Rica These authors discovered that barcoding raises the species count to 32 by revealing that each of the three parasitoid species previously considered as generalists actually are arrays of highly host specific cryptic species 63 For 15 morphospecies of polychaetes within the deep Antarctic benthos studied through DNA barcoding cryptic diversity was found in 50 of the cases Furthermore 10 previously overlooked morphospecies were detected increasing the total species richness in the sample by 233 64 nbsp Barcoding is a tool to vouch for food quality Here DNA from traditional Norwegian Christmas food is extracted at the molecular systematic lab at NTNU University Museum Diet analysis and food web application edit DNA barcoding and metabarcoding can be useful in diet analysis studies 65 and is typically used if prey specimens cannot be identified based on morphological characters 66 67 There is a range of sampling approaches in diet analysis DNA metabarcoding can be conducted on stomach contents 68 feces 67 69 saliva 70 or whole body analysis 50 71 In fecal samples or highly digested stomach contents it is often not possible to distinguish tissue from single species and therefore metabarcoding can be applied instead 67 72 Feces or saliva represent non invasive sampling approaches while whole body analysis often means that the individual needs to be killed first For smaller organisms sequencing for stomach content is then often done by sequencing the entire animal Barcoding for food safety edit DNA barcoding represents an essential tool to evaluate the quality of food products The purpose is to guarantee food traceability to minimize food piracy and to valuate local and typical agro food production Another purpose is to safeguard public health for example metabarcoding offers the possibility to identify groupers causing Ciguatera fish poisoning from meal remnants 73 or to separate poisonous mushrooms from edible ones Ref Biomonitoring and ecological assessment edit DNA barcoding can be used to assess the presence of endangered species for conservation efforts Ref or the presence of indicator species reflective to specific ecological conditions Ref for example excess nutrients or low oxygen levels Forensic Science edit DNA barcoding is often used for species identification in forensic science cases Unknown animal or plant samples at crime scenes can be found collected and identified in hopes of linking it to a suspect and getting a conviction 74 Poaching killing of endangered species and animal abuse are examples of crimes where DNA barcoding is used since animal DNA is often found 52 75 On the other hand plant DNA is usually used as trace evidence to link a suspect to a crime scene 76 Potentials and shortcomings editPotentials edit Traditional bioassessment methods are well established internationally and serve biomonitoring well as for example for aquatic bioassessment within the EU Directives WFD and MSFD However DNA barcoding could improve traditional methods for the following reasons DNA barcoding i can increase taxonomic resolution and harmonize the identification of taxa which are difficult to identify or lack experts ii can more accurately precisely relate environmental factors to specific taxa iii can increase comparability among regions iv allows for the inclusion of early life stages and fragmented specimens v allows delimitation of cryptic rare species vi allows for development of new indices e g rare cryptic species which may be sensitive tolerant to stressors vii increases the number of samples which can be processed and reduces processing time resulting in increased knowledge of species ecology viii is a non invasive way of monitoring when using eDNA methods 77 Time and cost edit DNA barcoding is faster than traditional morphological methods all the way from training through to taxonomic assignment It takes less time to gain expertise in DNA methods than becoming an expert in taxonomy In addition the DNA barcoding workflow i e from sample to result is generally quicker than traditional morphological workflow and allows the processing of more samples Taxonomic resolution edit DNA barcoding allows the resolution of taxa from higher e g family to lower e g species taxonomic levels that are otherwise too difficult to identify using traditional morphological methods like e g identification via microscopy For example Chironomidae the non biting midge are widely distributed in both terrestrial and freshwater ecosystems Their richness and abundance make them important for ecological processes and networks and they are one of many invertebrate groups used in biomonitoring Invertebrate samples can contain as many as 100 species of chironomids which often make up as much as 50 of a sample Despite this they are usually not identified below the family level because of the taxonomic expertise and time required 78 This may result in different chironomid species with different ecological preferences grouped together resulting in inaccurate assessment of water quality DNA barcoding provides the opportunity to resolve taxa and directly relate stressor effects to specific taxa such as individual chironomid species For example Beermann et al 2018 DNA barcoded Chironomidae to investigate their response to multiple stressors reduced flow increased fine sediment and increased salinity 79 After barcoding it was found that the chironomid sample consisted of 183 Operational Taxonomic Units OTUs i e barcodes sequences that are often equivalent to morphological species These 183 OTUs displayed 15 response types rather than the previously reported 80 two response types recorded when all chironomids were grouped together in the same multiple stressor study A similar trend was discovered in a study by Macher et al 2016 which discovered cryptic diversity within the New Zealand mayfly species Deleatidium sp This study found different response patterns of 12 molecular distinct OTUs to stressors which may change the consensus that this mayfly is sensitive to pollution 81 Shortcomings edit Despite the advantages offered by DNA barcoding it has also been suggested that DNA barcoding is best used as a complement to traditional morphological methods 77 This recommendation is based on multiple perceived challenges Physical parameters edit It is not completely straightforward to connect DNA barcodes with ecological preferences of the barcoded taxon in question as is needed if barcoding is to be used for biomonitoring For example detecting target DNA in aquatic systems depends on the concentration of DNA molecules at a site which in turn can be affected by many factors The presence of DNA molecules also depends on dispersion at a site e g direction or strength of currents It is not really known how DNA moves around in streams and lakes which makes sampling difficult Another factor might be the behavior of the target species e g fish can have seasonal changes of movements crayfish or mussels will release DNA in larger amounts just at certain times of their life moulting spawning For DNA in soil even less is known about distribution quantity or quality The major limitation of the barcoding method is that it relies on barcode reference libraries for the taxonomic identification of the sequences The taxonomic identification is accurate only if a reliable reference is available However most databases are still incomplete especially for smaller organisms e g fungi phytoplankton nematoda etc In addition current databases contain misidentifications spelling mistakes and other errors There is massive curation and completion effort around the databases for all organisms necessary involving large barcoding projects for example the iBOL project for the Barcode of Life Data Systems BOLD reference database 82 83 However completion and curation are difficult and time consuming Without vouchered specimens there can be no certainty about whether the sequence used as a reference is correct DNA sequence databases like GenBank contain many sequences that are not tied to vouchered specimens for example herbarium specimens cultured cell lines or sometimes images This is problematic in the face of taxonomic issues such as whether several species should be split or combined or whether past identifications were sound Reusing sequences not tied to vouchered specimens of initially misidentified organism may support incorrect conclusions and must be avoided 84 Therefore best practice for DNA barcoding is to sequence vouchered specimens 85 86 For many taxa it can be however difficult to obtain reference specimens for example with specimens that are difficult to catch available specimens are poorly conserved or adequate taxonomic expertise is lacking 84 Importantly DNA barcodes can also be used to create interim taxonomy in which case OTUs can be used as substitutes for traditional Latin binomials thus significantly reducing dependency on fully populated reference databases 87 Technological bias edit DNA barcoding also carries methodological bias from sampling to bioinformatics data analysis Beside the risk of contamination of the DNA sample by PCR inhibitors primer bias is one of the major sources of errors in DNA barcoding 88 89 The isolation of an efficient DNA marker and the design of primers is a complex process and considerable effort has been made to develop primers for DNA barcoding in different taxonomic groups 90 However primers will often bind preferentially to some sequences leading to differential primer efficiency and specificity and unrepresentative communities assessment and richness inflation 91 Thus the composition of the sample s communities sequences is mainly altered at the PCR step Besides PCR replication is often required but leads to an exponential increase in the risk of contamination Several studies have highlighted the possibility to use mitochondria enriched samples 92 93 or PCR free approaches to avoid these biases but as of today the DNA metabarcoding technique is still based on the sequencing of amplicons 90 Other bias enter the picture during the sequencing and during the bioinformatic processing of the sequences like the creation of chimeras Lack of standardization edit Even as DNA barcoding is more widely used and applied there is no agreement concerning the methods for DNA preservation or extraction the choices of DNA markers and primers set or PCR protocols The parameters of bioinformatics pipelines for example OTU clustering taxonomic assignment algorithms or thresholds etc are at the origin of much debate among DNA barcoding users 90 Sequencing technologies are also rapidly evolving together with the tools for the analysis of the massive amounts of DNA data generated and standardization of the methods is urgently needed to enable collaboration and data sharing at greater spatial and time scale This standardisation of barcoding methods at the European scale is part of the objectives of the European COST Action DNAqua net 94 and is also addressed by CEN the European Committee for Standardization 95 Another criticism of DNA barcoding is its limited efficiency for accurate discrimination below species level for example to distinguish between varieties for hybrid detection and that it can be affected by evolutionary rates citation needed Mismatches between conventional morphological and barcode based identification edit It is important to know that taxa lists derived by conventional morphological identification are not and maybe never will be directly comparable to taxa lists derived from barcode based identification because of several reasons The most important cause is probably the incompleteness and lack of accuracy of the molecular reference databases preventing a correct taxonomic assignment of eDNA sequences Taxa not present in reference databases will not be found by eDNA and sequences linked to a wrong name will lead to incorrect identification 77 Other known causes are a different sampling scale and size between a traditional and a molecular sample the possible analysis of dead organisms which can happen in different ways for both methods depending on organism group and the specific selection of identification in either method i e varying taxonomical expertise or possibility to identify certain organism groups respectively primer bias leading also to a potential biased analysis of taxa 77 Estimates of richness diversity edit DNA Barcoding can result in an over or underestimate of species richness and diversity Some studies suggest that artifacts identification of species not present in a community are a major cause of inflated biodiversity 96 97 The most problematic issue are taxa represented by low numbers of sequencing reads These reads are usually removed during the data filtering process since different studies suggest that most of these low frequency reads may be artifacts 98 However real rare taxa may exist among these low abundance reads 99 Rare sequences can reflect unique lineages in communities which make them informative and valuable sequences Thus there is a strong need for more robust bioinformatics algorithms that allow the differentiation between informative reads and artifacts Complete reference libraries would also allow a better testing of bioinformatics algorithms by permitting a better filtering of artifacts i e the removal of sequences lacking a counterpart among extant species and therefore it would be possible obtain a more accurate species assignment 100 Cryptic diversity can also result in inflated biodiversity as one morphological species may actually split into many distinct molecular sequences 77 This will go a long way in generating DNA reference data which is crucial for environmental DNA based biodiversity monitoring Megabarcoding editMegabarcoding is a term used to describe high throughput specimen based DNA barcoding where thousands of specimens can be barcoded simultaneously for species identification and discovery 101 102 103 104 105 nbsp Megabarcoding workflowThis is enabled by the use of third generation sequencing platforms including PacBio Sequel I II by Pacific Biosciences and MinION PromethION by Oxford Nanopore Technology As compared to Sanger sequencing megabarcoding is faster and cheaper allowing for the large scale generation of DNA barcodes for thousands of species 106 Applications edit Megabarcoding can help fill the dark taxa DNA barcode reference data gap for insects and accelerate species discovery 107 108 understand species diversity patterns 109 110 111 evaluate species richness 112 generate rapid biodiversity species inventories 113 track baseline shifts 114 and matching life history stages 115 Metabarcoding edit nbsp Differences in the standard methods for DNA barcoding and metabarcoding While DNA barcoding points to find a specific species metabarcoding looks for the whole community Main article Metabarcoding Metabarcoding is defined as the barcoding of DNA or eDNA environmental DNA that allows for simultaneous identification of many taxa within the same environmental sample however often within the same organism group The main difference between the approaches is that metabarcoding in contrast to barcoding does not focus on one specific organism but instead aims to determine species composition within a sample Methodology edit The metabarcoding procedure like general barcoding covers the steps of DNA extraction PCR amplification sequencing and data analysis 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 116 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 Applications edit Metabarcoding has the potential to complement biodiversity measures and even replace them in some instances especially as the technology advances and procedures gradually become cheaper more optimized and widespread 117 118 DNA metabarcoding applications include Biodiversity monitoring in terrestrial and aquatic environments Paleontology and ancient ecosystems Plant pollinator interactions Diet analysis and Food safety Advantages and challenges edit The general advantages and shortcomings for barcoding reviewed above are valid also for metabarcoding One particular drawback for metabarcoding studies is that there is no consensus yet regarding the optimal experimental design and bioinformatics criteria to be applied in eDNA metabarcoding 119 However there are current joined attempts like e g the EU COST network DNAqua Net to move forward by exchanging experience and knowledge to establish best practice standards for biomonitoring 77 Artificial DNA barcoding editIn 2014 researchers from ETH Zurich suggested using artificial sub micrometer sized DNA barcodes as an invisible oil tag The barcodes consist of synthetic DNA sequences inside magnetically recoverable silica particles They can be added to food oil in a very small amount down to 1 ppb as a label and can be retrieved at any time for authenticity test by PCR sequencing This method can be used to test olive oil for adulteration 120 See also editSubtopics Algae DNA barcoding 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Oxford ISBN 9780191079993 OCLC 1021883023 a href Template Cite book html title Template Cite book cite book a CS1 maint location missing publisher link Ruppert Krista M Kline Richard J Rahman Md Saydur January 2019 Past present and future perspectives of environmental DNA eDNA metabarcoding A systematic review in methods monitoring and applications of global eDNA Global Ecology and Conservation 17 e00547 doi 10 1016 j gecco 2019 e00547 Stoeck Thorsten Fruhe Larissa Forster Dominik Cordier Tristan Martins Catarina I M Pawlowski Jan February 2018 Environmental DNA metabarcoding of benthic bacterial communities indicates the benthic footprint of salmon aquaculture Marine Pollution Bulletin 127 139 149 doi 10 1016 j marpolbul 2017 11 065 PMID 29475645 Evans Darren M Kitson James J N Lunt David H Straw Nigel A Pocock Michael J O 2016 Merging DNA metabarcoding and ecological network analysis to understand and build resilient terrestrial ecosystems PDF Functional Ecology 30 12 1904 1916 doi 10 1111 1365 2435 12659 ISSN 1365 2435 Puddu M Paunescu D Stark W J Grass R N 2014 Magnetically Recoverable Thermostable Hydrophobic DNA Silica Encapsulates and Their Application as Invisible Oil Tags ACS Nano 8 3 2677 2685 doi 10 1021 nn4063853 PMID 24568212 External links editSweBOL FinBOL International Barcode of Life Project iBOL BOLD UNITE Diat barcode permanent dead link Retrieved from https en wikipedia org w index php title DNA barcoding amp oldid 1193494618, wikipedia, wiki, book, books, library,

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