DNA barcoding

DNA barcoding techniques were developed from early DNA sequencing work on microbial communities using the 5S rRNA gene.{{Cite journal |last1=Woese |first1=Carl R. |last2=Kandler |first2=Otto |last3=Wheelis |first3=Mark L. |date=1990 |title=Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya |journal=Proceedings of the National Academy of Sciences |volume=87 |issue=12 |pages=4576–4579 |url=https://www.pnas.org/content/pnas/87/12/4576.full.pdf?source=post_page--------------------------- |oclc=678728346 |doi=10.1073/pnas.87.12.4576|pmid=2112744 |pmc=54159 |bibcode=1990PNAS...87.4576W|doi-access=free }} 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.{{Cite journal|last1=Hebert|first1=Paul D. N.|last2=Cywinska|first2=Alina|last3=Ball|first3=Shelley L.|last4=deWaard|first4=Jeremy R.|date=2003-02-07|title=Biological identifications through DNA barcodes|journal=Proceedings of the Royal Society B: Biological Sciences|volume=270|issue=1512|pages=313–321|doi=10.1098/rspb.2002.2218|issn=1471-2954|pmc=1691236|pmid=12614582}} 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 as a suitable discriminatory tool between metazoan invertebrates.{{Cite journal|last1=Folmer|first1=O.|last2=Black|first2=M.|last3=Hoeh|first3=W.|last4=Lutz|first4=R.|last5=Vrijenhoek|first5=R.|date=October 1994|title=DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates|journal=Molecular Marine Biology and Biotechnology|volume=3|issue=5|pages=294–299|issn=1053-6426|pmid=7881515}} 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".

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.{{Citation|last1=Jelger Herder|title=Environmental DNA - a review of the possible applications for the detection of (invasive) species.}}

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.{{Cite journal|last1=Schrader|first1=C Then it can be this way because of DNA.|last2=Schielke|first2=A.|last3=Ellerbroek|first3=L.|last4=Johne|first4=R.|date=2012|title=PCR inhibitors – occurrence, properties and removal|journal=Journal of Applied Microbiology|volume=113|issue=5|pages=1014–1026|doi=10.1111/j.1365-2672.2012.05384.x|pmid=22747964|s2cid=30892831|issn=1365-2672|doi-access=free}} 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){{Cite journal|author1-link=Vincent Savolainen|last1=Savolainen|first1=Vincent|last2=Cowan|first2=Robyn S|last3=Vogler|first3=Alfried P|last4=Roderick|first4=George K|last5=Lane|first5=Richard|date=2005-10-29|title=Towards writing the encyclopaedia of life: an introduction to DNA barcoding|journal=Philosophical Transactions of the Royal Society B: Biological Sciences|volume=360|issue=1462|pages=1805–1811|doi=10.1098/rstb.2005.1730|pmid=16214739|pmc=1609222|issn=0962-8436}} 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.{{Cite journal|last=Piggott|first=Maxine P.|date=2016|title=Evaluating the effects of laboratory protocols on eDNA detection probability for an endangered freshwater fish|journal=Ecology and Evolution|volume=6|issue=9|pages=2739–2750|doi=10.1002/ece3.2083|issn=2045-7758|pmc=4798829|pmid=27066248}}{{Cite journal|last1=Ma|first1=Hongjuan|last2=Stewart|first2=Kathryn|last3=Lougheed|first3=Stephen|last4=Zheng|first4=Jinsong|last5=Wang|first5=Yuxiang|last6=Zhao|first6=Jianfu|date=2016|title=Characterization, optimization, and validation of environmental DNA (eDNA) markers to detect an endangered aquatic mammal|journal=Conservation Genetics Resources|volume=8|issue=4|pages=561–568|doi=10.1007/s12686-016-0597-9|s2cid=1613649|issn=1877-7252}}

File:HiSeq sequencers at SciLifeLab in Uppsala.jpg

When the DNA barcode marker region has been amplified, the next step is to sequence the marker region using DNA sequencing methods.{{Cite journal|last1=D’Amore|first1=Rosalinda|last2=Ijaz|first2=Umer Zeeshan|last3=Schirmer|first3=Melanie|last4=Kenny|first4=John G.|last5=Gregory|first5=Richard|last6=Darby|first6=Alistair C.|last7=Shakya|first7=Migun|last8=Podar|first8=Mircea|last9=Quince|first9=Christopher|date=2016-01-14|title=A comprehensive benchmarking study of protocols and sequencing platforms for 16S rRNA community profiling|journal=BMC Genomics|volume=17|issue=1|pages=55|doi=10.1186/s12864-015-2194-9|issn=1471-2164|pmc=4712552|pmid=26763898 |doi-access=free }} Many different sequencing platforms are available, and technical development is proceeding rapidly.

File:16S region variability.jpg

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 (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.{{Cite journal|last1=Kress|first1=W. J.|last2=Erickson|first2=D. L.|date=2008-02-26|title=DNA barcodes: Genes, genomics, and bioinformatics|journal=Proceedings of the National Academy of Sciences|volume=105|issue=8|pages=2761–2762|doi=10.1073/pnas.0800476105|issn=0027-8424|pmc=2268532|pmid=18287050|bibcode=2008PNAS..105.2761K|doi-access=free}}

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|date=February 2023}} or depending on the study question.

For animals, the most widely used barcode is mitochondrial cytochrome C oxidase I (COI) locus.{{Cite journal|last1=Hebert|first1=Paul D.N.|last2=Ratnasingham|first2=Sujeevan|last3=de Waard|first3=Jeremy R.|date=2003-08-07|title=Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species|journal=Proceedings of the Royal Society B: Biological Sciences|volume=270|issue=suppl_1|pages=S96-9|doi=10.1098/rsbl.2003.0025|issn=1471-2954|pmc=1698023|pmid=12952648}} 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.{{Cite journal|last=Blaxter|first=Mark L.|date=2004-04-29|editor-last=Godfray|editor-first=H. C. J.|editor2-last=Knapp|editor2-first=S.|title=The promise of a DNA taxonomy|journal=Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences|volume=359|issue=1444|pages=669–679|doi=10.1098/rstb.2003.1447|issn=1471-2970|pmc=1693355|pmid=15253352}} 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|date=February 2023}}

In plants, however, mitochondrial genes are not appropriate for DNA barcoding because they exhibit low mutation rates.{{Cite journal|last1=Fazekas|first1=Aron J.|last2=Burgess|first2=Kevin S.|last3=Kesanakurti|first3=Prasad R.|last4=Graham|first4=Sean W.|last5=Newmaster|first5=Steven G.|last6=Husband|first6=Brian C.|last7=Percy|first7=Diana M.|last8=Hajibabaei|first8=Mehrdad|last9=Barrett|first9=Spencer C. H.|date=2008-07-30|editor-last=DeSalle|editor-first=Robert|title=Multiple Multilocus DNA Barcodes from the Plastid Genome Discriminate Plant Species Equally Well|journal=PLOS ONE|volume=3|issue=7|pages=e2802|doi=10.1371/journal.pone.0002802|pmid=18665273|pmc=2475660|issn=1932-6203|bibcode=2008PLoSO...3.2802F|doi-access=free}} 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|date=February 2023}} The best discrimination between plant species has been achieved when using two or more chloroplast barcodes.{{Cite journal|last1=Kress|first1=W. John|last2=Erickson|first2=David L.|date=2007-06-06|editor-last=Shiu|editor-first=Shin-Han|title=A Two-Locus Global DNA Barcode for Land Plants: The Coding rbcL Gene Complements the Non-Coding trnH-psbA Spacer Region|journal=PLOS ONE|volume=2|issue=6|pages=e508|doi=10.1371/journal.pone.0000508|issn=1932-6203|pmc=1876818|pmid=17551588|bibcode=2007PLoSO...2..508K|doi-access=free}}

For bacteria, the small subunit of ribosomal RNA (16S) gene can be used for different taxa, as it is highly conserved.{{Cite journal|last1=Janda|first1=J. M.|last2=Abbott|first2=S. L.|date=2007-09-01|title=16S rRNA Gene Sequencing for Bacterial Identification in the Diagnostic Laboratory: Pluses, Perils, and Pitfalls|journal=Journal of Clinical Microbiology|volume=45|issue=9|pages=2761–2764|doi=10.1128/JCM.01228-07|issn=0095-1137|pmc=2045242|pmid=17626177}} Some studies suggest COI,{{Cite journal|last1=Smith|first1=M. Alex|last2=Bertrand|first2=Claudia|last3=Crosby|first3=Kate|last4=Eveleigh|first4=Eldon S.|last5=Fernandez-Triana|first5=Jose|last6=Fisher|first6=Brian L.|last7=Gibbs|first7=Jason|last8=Hajibabaei|first8=Mehrdad|last9=Hallwachs|first9=Winnie|date=2012-05-02|editor-last=Badger|editor-first=Jonathan H.|title=Wolbachia and DNA barcoding insects: Patterns, potential, and problems|journal=PLOS ONE|volume=7|issue=5|pages=e36514|doi=10.1371/journal.pone.0036514|issn=1932-6203|pmc=3342236|pmid=22567162|bibcode=2012PLoSO...736514S|doi-access=free}} type II chaperonin (cpn60){{Cite journal|last1=Links|first1=Matthew G.|last2=Dumonceaux|first2=Tim J.|last3=Hemmingsen|first3=Sean M.|last4=Hill|first4=Janet E.|date=2012-11-26|editor-last=Neufeld|editor-first=Josh|title=The Chaperonin-60 Universal Target Is a Barcode for Bacteria That Enables De Novo Assembly of Metagenomic Sequence Data|journal=PLOS ONE|volume=7|issue=11|pages=e49755|doi=10.1371/journal.pone.0049755|issn=1932-6203|pmc=3506640|pmid=23189159|bibcode=2012PLoSO...749755L|doi-access=free}} or β subunit of RNA polymerase (rpoB){{Cite journal|last1=Case|first1=R. J.|last2=Boucher|first2=Y.|last3=Dahllof|first3=I.|last4=Holmstrom|first4=C.|last5=Doolittle|first5=W. F.|last6=Kjelleberg|first6=S.|date=2007-01-01|title=Use of 16S rRNA and rpoB Genes as Molecular Markers for Microbial Ecology Studies|journal=Applied and Environmental Microbiology|volume=73|issue=1|pages=278–288|doi=10.1128/AEM.01177-06|issn=0099-2240|pmc=1797146|pmid=17071787|bibcode=2007ApEnM..73..278C }} also could serve as bacterial DNA barcodes.

Barcoding fungi is more challenging, and more than one primer combination might be required.{{Cite journal|last1=Bellemain|first1=Eva|last2=Carlsen|first2=Tor|last3=Brochmann|first3=Christian|last4=Coissac|first4=Eric|last5=Taberlet|first5=Pierre|last6=Kauserud|first6=Håvard|date=2010|title=ITS as an environmental DNA barcode for fungi: an in silico approach reveals potential PCR biases|journal=BMC Microbiology|volume=10|issue=1|pages=189|doi=10.1186/1471-2180-10-189|pmid=20618939|pmc=2909996|issn=1471-2180 |doi-access=free }} The COI marker performs well in certain fungi groups,{{Cite journal|last1=Seifert|first1=K. A.|last2=Samson|first2=R. A.|last3=deWaard|first3=J. R.|last4=Houbraken|first4=J.|last5=Levesque|first5=C. A.|last6=Moncalvo|first6=J.-M.|last7=Louis-Seize|first7=G.|last8=Hebert|first8=P. D. N.|date=2007-03-06|title=Prospects for fungus identification using CO1 DNA barcodes, with Penicillium as a test case|journal=Proceedings of the National Academy of Sciences|volume=104|issue=10|pages=3901–3906|doi=10.1073/pnas.0611691104|issn=0027-8424|pmc=1805696|pmid=17360450|doi-access=free}} but not equally well in others.{{Cite journal|last1=Dentinger|first1=Bryn T. M.|last2=Didukh|first2=Maryna Y.|last3=Moncalvo|first3=Jean-Marc|date=2011-09-22|editor-last=Schierwater|editor-first=Bernd|title=Comparing COI and ITS as DNA Barcode Markers for Mushrooms and Allies (Agaricomycotina)|journal=PLOS ONE|volume=6|issue=9|pages=e25081|doi=10.1371/journal.pone.0025081|issn=1932-6203|pmc=3178597|pmid=21966418|bibcode=2011PLoSO...625081D|doi-access=free}} Therefore, additional markers are being used, such as ITS rDNA and the large subunit of nuclear ribosomal RNA (28S LSU rRNA).{{Cite journal|last1=Khaund|first1=Polashree|last2=Joshi|first2=S.R.|date=October 2014|title=DNA barcoding of wild edible mushrooms consumed by the ethnic tribes of India|journal=Gene|volume=550|issue=1|pages=123–130|doi=10.1016/j.gene.2014.08.027|pmid=25130907}}

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|date=February 2023}}

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.{{Cite journal|last1=Weigand|first1=Hannah|last2=Beermann|first2=Arne J.|last3=Čiampor|first3=Fedor|last4=Costa|first4=Filipe O.|last5=Csabai|first5=Zoltán|last6=Duarte|first6=Sofia|last7=Geiger|first7=Matthias F.|last8=Grabowski|first8=Michał|last9=Rimet|first9=Frédéric|date=2019-03-14|title=DNA barcode reference libraries for the monitoring of aquatic biota in Europe: Gap-analysis and recommendations for future work|journal=bioRxiv|volume=678|pages=499–524|doi=10.1101/576553|pmid=31077928|bibcode=2019ScTEn.678..499W|hdl=11250/2608962|s2cid=92160002|hdl-access=free}} In the microbial world, there is no DNA information for most species names, and many DNA sequences cannot be assigned to any Linnaean binomial.Gottschling M, J Chacón, A Žerdoner Čalasan, St Neuhaus, J Kretschmann, H Stibor & U John (2020): Phylogenetic placement of environmental sequences using taxonomically reliable databases helps to rigorously assess dinophyte biodiversity in Bavarian lakes (Germany). Freshw Biol 65: 193–208. {{doi|10.1111/fwb.13413}} 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).{{Citation|last=Rdmpage|title=International Barcode of Life project (iBOL)|date=2016|url=http://www.gbif.org/dataset/040c5662-da76-4782-a48e-cdea1892d14c|publisher=Institute of Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow|doi=10.15468/inygc6|access-date=2019-05-14|type=Data Set}}

Launched in 2007, the Barcode of Life Data System (BOLD){{Cite journal|last1=Ratnasingham|first1=Sujeevan|last2=Hebert|first2=Paul D. N.|date=2007-01-24|title=BARCODING: bold: The Barcode of Life Data System: BARCODING|journal=Molecular Ecology Notes|volume=7|issue=3|pages=355–364|doi=10.1111/j.1471-8286.2007.01678.x|pmc=1890991|pmid=18784790}} 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.

The UNITE database{{Cite journal|last1=Nilsson|first1=Rolf Henrik|last2=Larsson|first2=Karl-Henrik|last3=Taylor|first3=Andy F. S.|last4=Bengtsson-Palme|first4=Johan|last5=Jeppesen|first5=Thomas S.|last6=Schigel|first6=Dmitry|last7=Kennedy|first7=Peter|last8=Picard|first8=Kathryn|last9=Glöckner|first9=Frank Oliver|date=2019-01-08|title=The UNITE database for molecular identification of fungi: handling dark taxa and parallel taxonomic classifications|journal=Nucleic Acids Research|volume=47|issue=D1|pages=D259–D264|doi=10.1093/nar/gky1022|issn=0305-1048|pmc=6324048|pmid=30371820}} 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{{Cite journal|last1=Rimet|first1=Frederic|last2=Gusev|first2=Evgenuy|last3=Kahlert|first3=Maria|last4=Kelly|first4=Martyn|last5=Kulikovskiy|first5=Maxim|last6=Maltsev|first6=Yevhen|last7=Mann|first7=David|last8=Pfannkuchen|first8=Martin|last9=Trobajo|first9=Rosa|date=2019-02-14|title=Diat.barcode, an open-access barcode library for diatoms|doi=10.15454/TOMBYZ|url=https://data.inrae.fr/dataset.xhtml?persistentId=doi:10.15454/TOMBYZ|website=data.inrae.fr|type=Data Set|publisher=Portail Data Inra}} database was first published under the name R-syst::diatom{{Cite journal|last1=Rimet|first1=Frédéric|last2=Chaumeil|first2=Philippe|last3=Keck|first3=François|last4=Kermarrec|first4=Lenaïg|last5=Vasselon|first5=Valentin|last6=Kahlert|first6=Maria|last7=Franc|first7=Alain|last8=Bouchez|first8=Agnès|date=2016|title=R-Syst::diatom: an open-access and curated barcode database for diatoms and freshwater monitoring|journal=Database|volume=2016|pages=baw016|doi=10.1093/database/baw016|issn=1758-0463|pmc=4795936|pmid=26989149}} 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.).

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.

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,{{Cite journal|title=Introducing mothur : open-source, platform-independent, community-supported software for describing and comparing microbial communities|author1=Schloss, Patrick D. |author2=Westcott, Sarah L. |author3=Ryabin, Thomas |author4=Hall, Justine R. |author5=Hartmann, Martin |author6=Hollister, Emily B. |author7=Lesniewski, Ryan A. |author8=Oakley, Brian B. |author9=Parks, Donovan H. |author10=Robinson, Courtney J. |author11=Sahl, Jason W. |author12=Stres, Blaž. |author13=Thallinger, Gerhard G. |author14=Horn, David J. |author15=van. Weber, Caroly F.|journal=Applied and Environmental Microbiology |year=2009 |volume=75 |issue=23 |pages=7537–41 |doi=10.1128/AEM.01541-09 |pmid=19801464 |pmc=2786419 |bibcode=2009ApEnM..75.7537S |oclc=780918718}} Uparse,{{Cite journal|last=Edgar|first=Robert C|date=2013-08-18|title=UPARSE: highly accurate OTU sequences from microbial amplicon reads|journal=Nature Methods|volume=10|issue=10|pages=996–998|doi=10.1038/nmeth.2604|pmid=23955772|s2cid=7181682|issn=1548-7091}} Qiime,{{Cite journal|last1=Caporaso|first1=J Gregory|last2=Kuczynski|first2=Justin|last3=Stombaugh|first3=Jesse|last4=Bittinger|first4=Kyle|last5=Bushman|first5=Frederic D|last6=Costello|first6=Elizabeth K|last7=Fierer|first7=Noah|last8=Peña|first8=Antonio Gonzalez|last9=Goodrich|first9=Julia K|date=May 2010|title=QIIME allows analysis of high-throughput community sequencing data|journal=Nature Methods|volume=7|issue=5|pages=335–336|doi=10.1038/nmeth.f.303|issn=1548-7091|pmc=3156573|pmid=20383131}} Galaxy,{{Cite journal|last1=Afgan|first1=Enis|last2=Baker|first2=Dannon|last3=Batut|first3=Bérénice|last4=van den Beek|first4=Marius|last5=Bouvier|first5=Dave|last6=Čech|first6=Martin|last7=Chilton|first7=John|last8=Clements|first8=Dave|last9=Coraor|first9=Nate|date=2018-07-02|title=The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update|journal=Nucleic Acids Research|volume=46|issue=W1|pages=W537–W544|doi=10.1093/nar/gky379|issn=0305-1048|pmc=6030816|pmid=29790989}} Obitools,{{Cite journal|last1=Boyer|first1=Frédéric|last2=Mercier|first2=Céline|last3=Bonin|first3=Aurélie|last4=Le Bras|first4=Yvan|last5=Taberlet|first5=Pierre|last6=Coissac|first6=Eric|date=2015-05-26|title=obitools: aunix-inspired software package for DNA metabarcoding|journal=Molecular Ecology Resources|volume=16|issue=1|pages=176–182|doi=10.1111/1755-0998.12428|pmid=25959493|s2cid=39412858|issn=1755-098X}} JAMP,{{Citation|last=Elbrecht|first=Vasco|title=GitHub - VascoElbrecht/JAMP: JAMP: Just Another Metabarcoding Pipeline.|date=2019-04-30|url=https://github.com/VascoElbrecht/JAMP|access-date=2019-05-14}} Barque,{{Citation|last=Normandeau|first=Eric|title=GitHub - enormandeau/barque: Barque: Environmental DNA metabarcoding analysis.|date=2020-01-21|url=https://github.com/enormandeau/barque|access-date=2020-01-21}} and DADA2.{{Cite journal|last1=Callahan|first1=Benjamin J|last2=McMurdie|first2=Paul J|last3=Rosen|first3=Michael J|last4=Han|first4=Andrew W|last5=Johnson|first5=Amy Jo A|last6=Holmes|first6=Susan P|date=July 2016|title=DADA2: High-resolution sample inference from Illumina amplicon data|journal=Nature Methods|volume=13|issue=7|pages=581–583|doi=10.1038/nmeth.3869|issn=1548-7091|pmc=4927377|pmid=27214047}}

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.{{Cite journal|title=Waste Not, Want Not: Why Rarefying Microbiome Data is Inadmissible|journal=PLOS Computational Biology|volume=10|issue=4|pages=e1003531|doi=10.1371/journal.pcbi.1003531|pmid=24699258|pmc=3974642|year=2014|last1=McMurdie|first1=Paul J.|last2=Holmes|first2=Susan|bibcode=2014PLSCB..10E3531M|arxiv=1310.0424 |doi-access=free }}

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.{{Cite journal|last1=Valiente|first1=Gabriel|last2=Jansson|first2=Jesper|last3=Clemente|first3=Jose Carlos|last4=Alonso-Alemany|first4=Daniel|date=2011-10-10|title=Taxonomic Assignment in Metagenomics with TANGO|journal=EMBnet.journal|volume=17|issue=2|pages=16–20|doi=10.14806/ej.17.2.237|issn=2226-6089|doi-access=free|hdl=2117/16286|hdl-access=free}} 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).

In some cases, specimens with identical (COI) DNA barcodes clearly belong to different species, e.g. species of the fish genus Chromis.{{Cite journal |last1=Pyle |first1=Richard L. |last2=Earle |first2=John L. |last3=Greene |first3=Brian D. |date=2008-01-01 |title=Five new species of the damselfish genus Chromis (Perciformes: Labroidei: Pomacentridae) from deep coral reefs in the tropical western Pacific |url=https://mapress.com/zt/article/view/zootaxa.1671.1.2 |journal=Zootaxa |volume=1671 |issue=1 |doi=10.11646/zootaxa.1671.1.2 |issn=1175-5334}}

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,{{Cite journal|last1=Schnell|first1=Ida Bærholm|last2=Thomsen|first2=Philip Francis|last3=Wilkinson|first3=Nicholas|last4=Rasmussen|first4=Morten|last5=Jensen|first5=Lars R.D.|last6=Willerslev|first6=Eske|last7=Bertelsen|first7=Mads F.|last8=Gilbert|first8=M. Thomas P.|date=April 2012|title=Screening mammal biodiversity using DNA from leeches|journal=Current Biology|volume=22|issue=8|pages=R262–R263|doi=10.1016/j.cub.2012.02.058|pmid=22537625|s2cid=18058748|doi-access=free}} linking egg and larval stages to adult species, securing intellectual property rights for bioresources, framing global management plans for conservation strategies, elucidate feeding niches,{{Cite book|title=DNA Barcoding in Marine Perspectives : Assessment and Conservation of Biodiversity.|last=Subrata.|first=Trivedi|date=2016|publisher=Springer International Publishing|others=Ansari, Abid Ali., Ghosh, Sankar K., Rehman, Hasibur.|isbn=9783319418407|location=Cham|oclc=958384953}} and forensic science.{{Cite journal |last1=Dalton |first1=Desiré Lee |last2=de Bruyn |first2=Marli |last3=Thompson |first3=Tia |last4=Kotzé |first4=Antoinette |date=2020-12-01 |title=Assessing the utility of DNA barcoding in wildlife forensic cases involving South African antelope |journal=Forensic Science International: Reports |language=en |volume=2 |pages=100071 |doi=10.1016/j.fsir.2020.100071 |s2cid=213926390 |issn=2665-9107|doi-access=free }} 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.

Specific short DNA sequences or markers from a standardized region of the genome can provide a DNA barcode for identifying species.{{Cite journal|last1=Hebert|first1=Paul D. N.|last2=Stoeckle|first2=Mark Y.|last3=Zemlak|first3=Tyler S.|last4=Francis|first4=Charles M.|date=October 2004|title=Identification of Birds through DNA Barcodes|journal=PLOS Biology|volume=2|issue=10|pages=e312|doi=10.1371/journal.pbio.0020312|issn=1545-7885|pmc=518999|pmid=15455034 |doi-access=free }} 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.{{Cite journal|last1=Costa|first1=Filipe O|last2=Carvalho|first2=Gary R|date=December 2007|title=The Barcode of Life Initiative: synopsis and prospective societal impacts of DNA barcoding of Fish|journal=Genomics, Society and Policy|volume=3|issue=2|pages=29|doi=10.1186/1746-5354-3-2-29|issn=1746-5354|pmc=5425017 |doi-access=free }} 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.{{Cite journal|last1=Lahaye|first1=R.|last2=van der Bank|first2=M.|last3=Bogarin|first3=D.|last4=Warner|first4=J.|last5=Pupulin|first5=F.|last6=Gigot|first6=G.|last7=Maurin|first7=O.|last8=Duthoit|first8=S.|last9=Barraclough|first9=T. G.|date=2008-02-26|title=DNA barcoding the floras of biodiversity hotspots|journal=Proceedings of the National Academy of Sciences|volume=105|issue=8|pages=2923–2928|doi=10.1073/pnas.0709936105|issn=0027-8424|pmc=2268561|pmid=18258745|doi-access=free}}

Alien species can be detected via barcoding.{{Cite journal|last1=Xu|first1=Song-Zhi|last2=Li|first2=Zhen-Yu|last3=Jin|first3=Xiao-Hua|date=January 2018|title=DNA barcoding of invasive plants in China: A resource for identifying invasive plants|journal=Molecular Ecology Resources|volume=18|issue=1|pages=128–136|doi=10.1111/1755-0998.12715|pmid=28865184|s2cid=24911390}}{{Cite journal|last1=Liu|first1=Junning|last2=Jiang|first2=Jiamei|last3=Song|first3=Shuli|last4=Tornabene|first4=Luke|last5=Chabarria|first5=Ryan|last6=Naylor|first6=Gavin J. P.|last7=Li|first7=Chenhong|date=December 2017|title=Multilocus DNA barcoding – Species Identification with Multilocus Data|journal=Scientific Reports|volume=7|issue=1|pages=16601|doi=10.1038/s41598-017-16920-2|issn=2045-2322|pmc=5709489|pmid=29192249|bibcode=2017NatSR...716601L}} 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 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.{{Cite journal|last1=Nagoshi|first1=Rodney N.|last2=Brambila|first2=Julieta|last3=Meagher|first3=Robert L.|date=November 2011|title=Use of DNA barcodes to identify invasive armyworm Spodoptera species in Florida|journal=Journal of Insect Science|volume=11|issue=154|pages=154|doi=10.1673/031.011.15401|issn=1536-2442|pmc=3391933|pmid=22239735}} The high efficiency of DNA identification is shown relative to the traditional monitoring of biological invasions.{{Cite journal |last1=Karabanov |first1=D.P. |last2=Bekker |first2=E.I. |last3=Pavlov |first3=D.D. |last4=Borovikova |first4=E.A. |last5=Kodukhova |first5=Y.V. |last6=Kotov |first6=A.A. |date=1 February 2022 |title=New Sets of Primers for DNA Identification of Non-Indigenous Fish Species in the Volga-Kama Basin (European Russia) |journal=Water |volume=14 |issue=3 |pages=437 |issn=2073-4441 |doi=10.3390/w14030437 |doi-access=free}}

DNA barcoding enables the identification and recognition of cryptic species.{{Cite journal|last1=Thongtam na Ayudhaya|first1=Pradipunt|last2=Muangmai|first2=Narongrit|last3=Banjongsat|first3=Nuwadee|last4=Singchat|first4=Worapong|last5=Janekitkarn|first5=Sommai|last6=Peyachoknagul|first6=Surin|last7=Srikulnath|first7=Kornsorn|date=June 2017|title=Unveiling cryptic diversity of the anemonefish genera Amphiprion and Premnas (Perciformes: Pomacentridae) in Thailand with mitochondrial DNA barcodes|journal=Agriculture and Natural Resources|volume=51|issue=3|pages=198–205|doi=10.1016/j.anres.2017.07.001|doi-access=free}} 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.{{Cite journal|last1=Hebert|first1=P. D. N.|last2=Penton|first2=E. H.|last3=Burns|first3=J. M.|last4=Janzen|first4=D. H.|last5=Hallwachs|first5=W.|date=2004-10-12|title=Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator|journal=Proceedings of the National Academy of Sciences|volume=101|issue=41|pages=14812–14817|doi=10.1073/pnas.0406166101|issn=0027-8424|pmc=522015|pmid=15465915|bibcode=2004PNAS..10114812H|doi-access=free}} 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.{{Cite journal|last=Brower|first=Andrew V.Z.|date=June 2006|title=Problems with DNA barcodes for species delimitation: 'Ten species' of Astraptes fulgerator reassessed (Lepidoptera: Hesperiidae)|journal=Systematics and Biodiversity|volume=4|issue=2|pages=127–132|doi=10.1017/S147720000500191X|s2cid=54687052|issn=1477-2000}} 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.{{Cite journal|last1=Smith|first1=M. A.|last2=Woodley|first2=N. E.|last3=Janzen|first3=D. H.|last4=Hallwachs|first4=W.|last5=Hebert|first5=P. D. N.|date=2006-03-07|title=DNA barcodes reveal cryptic host-specificity within the presumed polyphagous members of a genus of parasitoid flies (Diptera: Tachinidae)|journal=Proceedings of the National Academy of Sciences|volume=103|issue=10|pages=3657–3662|doi=10.1073/pnas.0511318103|issn=0027-8424|pmc=1383497|pmid=16505365|doi-access=free}} 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%.{{Cite journal|last1=Brasier|first1=Madeleine J.|last2=Wiklund|first2=Helena|last3=Neal|first3=Lenka|last4=Jeffreys|first4=Rachel|last5=Linse|first5=Katrin|last6=Ruhl|first6=Henry|last7=Glover|first7=Adrian G.|date=November 2016|title=DNA barcoding uncovers cryptic diversity in 50% of deep-sea Antarctic polychaetes|journal=Royal Society Open Science|volume=3|issue=11|pages=160432|doi=10.1098/rsos.160432|issn=2054-5703|pmc=5180122|pmid=28018624|bibcode=2016RSOS....360432B}}

File:DNA-strekkoding av julemat - DNA barcoding of Christmas food (16008786266).jpg

DNA barcoding and metabarcoding can be useful in diet analysis studies,{{Cite journal|last1=Pompanon|first1=Francois|last2=Deagle|first2=Bruce E.|last3=Symondson|first3=William O. C.|last4=Brown|first4=David S.|last5=Jarman|first5=Simon N.|last6=Taberlet|first6=Pierre|date=April 2012|title=Who is eating what: diet assessment using next generation sequencing: NGS DIET ANALYSIS|journal=Molecular Ecology|volume=21|issue=8|pages=1931–1950|doi=10.1111/j.1365-294X.2011.05403.x|pmid=22171763|s2cid=10013333|doi-access=free}} and is typically used if prey specimens cannot be identified based on morphological characters.{{Cite journal|last1=Valentini|first1=Alice|last2=Pompanon|first2=François|last3=Taberlet|first3=Pierre|date=February 2009|title=DNA barcoding for ecologists|journal=Trends in Ecology & Evolution|volume=24|issue=2|pages=110–117|doi=10.1016/j.tree.2008.09.011|pmid=19100655}}{{Cite journal|last1=Kaunisto|first1=Kari M.|last2=Roslin|first2=Tomas|last3=Sääksjärvi|first3=Ilari E.|last4=Vesterinen|first4=Eero J.|date=October 2017|title=Pellets of proof: First glimpse of the dietary composition of adult odonates as revealed by metabarcoding of feces|journal=Ecology and Evolution|volume=7|issue=20|pages=8588–8598|doi=10.1002/ece3.3404|pmc=5648679|pmid=29075474}} There is a range of sampling approaches in diet analysis: DNA metabarcoding can be conducted on stomach contents,{{Cite journal|last1=Harms-Tuohy|first1=Ca|last2=Schizas|first2=Nv|last3=Appeldoorn|first3=Rs|date=2016-10-25|title=Use of DNA metabarcoding for stomach content analysis in the invasive lionfish Pterois volitans in Puerto Rico|journal=Marine Ecology Progress Series|volume=558|pages=181–191|doi=10.3354/meps11738|issn=0171-8630|bibcode=2016MEPS..558..181H|doi-access=free}} feces,{{Cite journal|last1=Kowalczyk|first1=Rafał|last2=Taberlet|first2=Pierre|last3=Coissac|first3=Eric|last4=Valentini|first4=Alice|last5=Miquel|first5=Christian|last6=Kamiński|first6=Tomasz|last7=Wójcik|first7=Jan M.|date=February 2011|title=Influence of management practices on large herbivore diet—Case of European bison in Białowieża Primeval Forest (Poland)|journal=Forest Ecology and Management|volume=261|issue=4|pages=821–828|doi=10.1016/j.foreco.2010.11.026}} saliva{{Cite journal|last1=Nichols|first1=Ruth V.|last2=Cromsigt|first2=Joris P. G. M.|last3=Spong|first3=Göran|date=December 2015|title=Using eDNA to experimentally test ungulate browsing preferences|journal=SpringerPlus|volume=4|issue=1|pages=489|doi=10.1186/s40064-015-1285-z|issn=2193-1801|pmc=4565800|pmid=26380165 |doi-access=free }} or whole body analysis.{{Cite journal|last1=Agusti|first1=N.|last2=Shayler|first2=S. P.|last3=Harwood|first3=J. D.|last4=Vaughan|first4=I. P.|last5=Sunderland|first5=K. D.|last6=Symondson|first6=W. O. C.|date=December 2003|title=Collembola as alternative prey sustaining spiders in arable ecosystems: prey detection within predators using molecular markers|journal=Molecular Ecology|volume=12|issue=12|pages=3467–3475|doi=10.1046/j.1365-294X.2003.02014.x|pmid=14629361|s2cid=7985256|issn=0962-1083|doi-access=free}} 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.{{Cite journal|last1=Valentini|first1=Alice|last2=Miquel|first2=Christian|last3=Nawaz|first3=Muhammad Ali|last4=Bellemain|first4=Eva|last5=Coissac|first5=Eric|last6=Pompanon|first6=François|last7=Gielly|first7=Ludovic|last8=Cruaud|first8=Corinne|last9=Nascetti|first9=Giuseppe|date=January 2009|title=New perspectives in diet analysis based on DNA barcoding and parallel pyrosequencing: the trn L approach|journal=Molecular Ecology Resources|volume=9|issue=1|pages=51–60|doi=10.1111/j.1755-0998.2008.02352.x|pmid=21564566|s2cid=5308081|doi-access=free}} 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.

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,{{Cite journal|last1=Friedman|first1=Melissa|last2=Fernandez|first2=Mercedes|last3=Backer|first3=Lorraine|last4=Dickey|first4=Robert|last5=Bernstein|first5=Jeffrey|last6=Schrank|first6=Kathleen|last7=Kibler|first7=Steven|last8=Stephan|first8=Wendy|last9=Gribble|first9=Matthew|date=2017-03-14|title=An Updated Review of Ciguatera Fish Poisoning: Clinical, Epidemiological, Environmental, and Public Health Management|journal=Marine Drugs|volume=15|issue=3|pages=72|doi=10.3390/md15030072|issn=1660-3397|pmc=5367029|pmid=28335428|doi-access=free}} or to separate poisonous mushrooms from edible ones (Ref).

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.

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.{{Cite journal |date=2018-03-04 |title=Illegal product manufacturing and exportation from Pakistan: Revealing the factuality of highly processed wildlife skin samples via DNA mini-barcoding |url=http://resolver.scholarsportal.info/resolve/15257770/v37i0003/179_ipmaefwssvdm.xml |journal=Nucleosides, Nucleotides and Nucleic Acids |volume=37 |issue=3 |pages=179–185|doi=10.1080/15257770.2018.1450507 |pmid=29608392 |last1=Khan |first1=F. M. |last2=William |first2=K. |last3=Aruge |first3=S. |last4=Janjua |first4=S. |last5=Shah |first5=S. A. |s2cid=4623232 }} Poaching, killing of endangered species, and animal abuse are examples of crimes where DNA barcoding is used, since animal DNA is often found.{{Cite journal |last1=Mwale |first1=Monica |last2=Dalton |first2=Desire L. |last3=Jansen |first3=Raymond |last4=De Bruyn |first4=Marli |last5=Pietersen |first5=Darren |last6=Mokgokong |first6=Prudent S. |last7=Kotzé |first7=Antoinette |date=March 2017 |editor-last=Steinke |editor-first=Dirk |title=Forensic application of DNA barcoding for identification of illegally traded African pangolin scales |url=http://www.nrcresearchpress.com/doi/10.1139/gen-2016-0144 |journal=Genome |language=en |volume=60 |issue=3 |pages=272–284 |doi=10.1139/gen-2016-0144 |pmid=28177847 |hdl=1807/75671 |s2cid=207093202 |issn=0831-2796|hdl-access=free }} On the other hand, plant DNA is usually used as trace evidence to link a suspect to a crime scene.{{Cite journal |last1=Liu |first1=Yanlei |last2=Xu |first2=Chao |last3=Dong |first3=Wenpan |last4=Yang |first4=Xueying |last5=Zhou |first5=Shiliang |date=2021-07-01 |title=Determination of a criminal suspect using environmental plant DNA metabarcoding technology |url=https://www.sciencedirect.com/science/article/pii/S0379073821001481 |journal=Forensic Science International |language=en |volume=324 |pages=110828 |doi=10.1016/j.forsciint.2021.110828 |pmid=34000616 |s2cid=234768561 |issn=0379-0738}}

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.{{Cite journal|last1=Pawlowski|first1=Jan|last2=Kelly-Quinn|first2=Mary|last3=Altermatt|first3=Florian|last4=Apothéloz-Perret-Gentil|first4=Laure|last5=Beja|first5=Pedro|last6=Boggero|first6=Angela|last7=Borja|first7=Angel|last8=Bouchez|first8=Agnès|last9=Cordier|first9=Tristan|date=2018|title=The future of biotic indices in the ecogenomic era: Integrating (e)DNA metabarcoding in biological assessment of aquatic ecosystems|journal=Science of the Total Environment|volume=637–638|pages=1295–1310|doi=10.1016/j.scitotenv.2018.05.002|pmid=29801222|bibcode=2018ScTEn.637.1295P|doi-access=free|hdl=20.500.12327/138|hdl-access=free}}

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.

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.{{Cite book|title=The Chironomidae|date=1995|publisher=Springer Netherlands|isbn=9789401043083|editor-last=Armitage|editor-first=Patrick D.|location=Dordrecht|doi=10.1007/978-94-011-0715-0|s2cid=46138170|editor-last2=Cranston|editor-first2=Peter S.|editor-last3=Pinder|editor-first3=L. C. V.}} 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.{{Cite journal|last1=Beermann|first1=Arne J.|last2=Zizka|first2=Vera M. A.|last3=Elbrecht|first3=Vasco|last4=Baranov|first4=Viktor|last5=Leese|first5=Florian|date=2018-07-24|title=DNA metabarcoding reveals the complex and hidden responses of chironomids to multiple stressors|journal=Environmental Sciences Europe|volume=30|issue=1|pages=26|doi=10.1186/s12302-018-0157-x|s2cid=51802465|issn=2190-4715|doi-access=free}} 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 {{Cite journal|last1=Beermann|first1=Arne J.|last2=Elbrecht|first2=Vasco|last3=Karnatz|first3=Svenja|last4=Ma|first4=Li|last5=Matthaei|first5=Christoph D.|last6=Piggott|first6=Jeremy J.|last7=Leese|first7=Florian|date=2018|title=Multiple-stressor effects on stream macroinvertebrate communities: A mesocosm experiment manipulating salinity, fine sediment and flow velocity|journal=Science of the Total Environment|volume=610–611|pages=961–971|doi=10.1016/j.scitotenv.2017.08.084|pmid=28830056|bibcode=2018ScTEn.610..961B}} 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 [http://www.terrain.net.nz/friends-of-te-henui-group/invertebrates-freshwater-new-zealand/mayfly-nymph-genus-deleatidium.html 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.{{Cite journal|last1=Macher|first1=Jan N.|last2=Salis|first2=Romana K.|last3=Blakemore|first3=Katie S.|last4=Tollrian|first4=Ralph|last5=Matthaei|first5=Christoph D.|last6=Leese|first6=Florian|date=2016|title=Multiple-stressor effects on stream invertebrates: DNA barcoding reveals contrasting responses of cryptic mayfly species|journal=Ecological Indicators|volume=61|pages=159–169|doi=10.1016/j.ecolind.2015.08.024}}

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. This recommendation is based on multiple perceived challenges.

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).{{Cite web|url=http://ibol.org/|title=The International Barcode of Life Consortium|website=International Barcode of Life|access-date=2019-03-29}}{{Cite web|url=http://www.boldsystems.org/|title=Bold Systems v4|website=www.boldsystems.org|access-date=2019-04-02}} 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.{{cite journal|last1=Ogwang |first1=Joel|last2=Bariche|first2=Michel |last3=Bos|first3=Arthur R.|date=2020|title= Genetic Diversity and Phylogenetic Relationships of Threadfin Breams (Nemipterus spp.) from the Red Sea and eastern Mediterranean Sea|journal=Genome|volume=63|issue=3|pages=207–216|doi= 10.1139/gen-2019-0163|pmid=32678985|doi-access=free}} Therefore, best practice for DNA barcoding is to sequence vouchered specimens.{{Cite journal|last1=Schander|first1=Christoffer|last2=Willassen|first2=Endre|date=2005|title=What can biological barcoding do for marine biology?|journal=Marine Biology Research|volume=1|issue=1|pages=79–83|doi=10.1080/17451000510018962|s2cid=84070971|issn=1745-1000|doi-access=free}}{{Cite journal|last=Miller|first=S. E.|date=2007-03-20|title=DNA barcoding and the renaissance of taxonomy|journal=Proceedings of the National Academy of Sciences|volume=104|issue=12|pages=4775–4776|doi=10.1073/pnas.0700466104|issn=0027-8424|pmc=1829212|pmid=17363473|bibcode=2007PNAS..104.4775M|doi-access=free}} 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.

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.{{Cite journal|last=Ratnasingham|first=S.|date=2013|title=A DNA-based registry for all animal species: the Barcode Index Number (BIN) system|journal=PLOS ONE|volume=8|issue=7|pages=e66213|doi=10.1371/journal.pone.0066213|pmid=23861743|pmc=3704603|bibcode=2013PLoSO...866213R|doi-access=free}}

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.{{Cite journal|last1=Leese|first1=Florian|last2=Elbrecht|first2=Vasco|date=2015-07-08|title=Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol|journal=PLOS ONE|volume=10|issue=7|pages=e0130324|doi=10.1371/journal.pone.0130324|issn=1932-6203|pmc=4496048|pmid=26154168|bibcode=2015PLoSO..1030324E|doi-access=free}}{{Cite journal|last1=Elbrecht|first1=Vasco|last2=Vamos|first2=Ecaterina Edith|last3=Meissner|first3=Kristian|last4=Aroviita|first4=Jukka|last5=Leese|first5=Florian|date=2017|title=Assessing strengths and weaknesses of DNA metabarcoding-based macroinvertebrate identification for routine stream monitoring|journal=Methods in Ecology and Evolution|volume=8|issue=10|pages=1265–1275|doi=10.1111/2041-210X.12789|issn=2041-210X|doi-access=free}} 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.{{Cite journal|title=The future of biotic indices in the ecogenomic era: Integrating (E)DNA metabarcoding in biological assessment of aquatic ecosystems|pages=1295–1310|journal=Science of the Total Environment|volume=637–638|doi=10.1016/j.scitotenv.2018.05.002|pmid=29801222|date=October 2018|last1=Pawlowski|first1=J.|last2=Kelly-Quinn|first2=M.|last3=Altermatt|first3=F.|last4=Apothéloz-Perret-Gentil|first4=L.|last5=Beja|first5=P.|last6=Boggero|first6=A.|last7=Borja|first7=A.|last8=Bouchez|first8=A.|last9=Cordier|first9=T.|last10=Domaizon|first10=I.|last11=Feio|first11=M. J.|last12=Filipe|first12=A. F.|last13=Fornaroli|first13=R.|last14=Graf|first14=W.|last15=Herder|first15=J.|last16=Van Der Hoorn|first16=B.|last17=Iwan Jones|first17=J.|last18=Sagova-Mareckova|first18=M.|last19=Moritz|first19=C.|last20=Barquín|first20=J.|last21=Piggott|first21=J. J.|last22=Pinna|first22=M.|last23=Rimet|first23=F.|last24=Rinkevich|first24=B.|last25=Sousa-Santos|first25=C.|last26=Specchia|first26=V.|last27=Trobajo|first27=R.|last28=Vasselon|first28=V.|last29=Vitecek|first29=S.|last30=Zimmerman|first30=J.|display-authors=29|bibcode=2018ScTEn.637.1295P|doi-access=free|hdl=20.500.12327/138|hdl-access=free}} However, primers will often bind preferentially to some sequences, leading to differential primer efficiency and specificity and unrepresentative communities’ assessment and richness inflation.{{Cite journal|last1=Quince|first1=Christopher|last2=Sloan|first2=William T.|last3=Hall|first3=Neil|last4=D'Amore|first4=Rosalinda|last5=Ijaz|first5=Umer Z.|last6=Schirmer|first6=Melanie|date=2015-03-31|title=Insight into biases and sequencing errors for amplicon sequencing with the Illumina MiSeq platform|journal=Nucleic Acids Research|volume=43|issue=6|pages=e37|doi=10.1093/nar/gku1341|issn=0305-1048|pmc=4381044|pmid=25586220}} 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 {{Cite journal|last1=Huang|first1=Quanfei|last2=Li|first2=Jiguang|last3=Fu|first3=Ribei|last4=Tang|first4=Min|last5=Zhou|first5=Lili|last6=Su|first6=Xu|last7=Yang|first7=Qing|last8=Liu|first8=Shanlin|last9=Li|first9=Yiyuan|date=2013-12-01|title=Ultra-deep sequencing enables high-fidelity recovery of biodiversity for bulk arthropod samples without PCR amplification|journal=GigaScience|volume=2|issue=1|pages=4|doi=10.1186/2047-217X-2-4|pmc=3637469|pmid=23587339 |doi-access=free }}{{Cite journal|last1=Macher|first1=Jan-Niklas|last2=Zizka|first2=Vera Marie Alida|last3=Weigand|first3=Alexander Martin|last4=Leese|first4=Florian|date=2018|title=A simple centrifugation protocol for metagenomic studies increases mitochondrial DNA yield by two orders of magnitude|journal=Methods in Ecology and Evolution|volume=9|issue=4|pages=1070–1074|doi=10.1111/2041-210X.12937|issn=2041-210X|doi-access=free}} or PCR-free approaches to avoid these biases, but as of {{As of|2018|bare=yes}}, the DNA metabarcoding technique is still based on the sequencing of amplicons. Other biases enter the picture during the sequencing and during the bioinformatic processing of the sequences, like the creation of chimeras.

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. 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 {{Cite web|url=https://dnaqua.net/|title=DNAquaNet|access-date=2019-03-29}} and is also addressed by CEN (the European Committee for Standardization).CEN (2018) CEN/TC 230/WORKING GROUP 2 – Proposal for a new Working Group WG28 “DNA and eDNA methods” A plan to fulfil the DNA and eDNA standardization needs of EU legislation in Water Policy (Proposal following decisions of the 2017 Berlin Meeting of CEN/TC 230, its Working Groups and eDNA COST representatives)

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|date=July 2022}}.

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. 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.

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.{{Cite journal|last1=Sloan|first1=William T.|last2=Read|first2=L. Fiona|last3=Head|first3=Ian M.|last4=Neil Hall|last5=Davenport|first5=Russell J.|last6=Curtis|first6=Thomas P.|last7=Lanzén|first7=Anders|last8=Quince|first8=Christopher|date=2009|title=Accurate determination of microbial diversity from 454 pyrosequencing data|journal=Nature Methods|volume=6|issue=9|pages=639–641|doi=10.1038/nmeth.1361|pmid=19668203|hdl=1956/6529|s2cid=1975660|issn=1548-7105|hdl-access=free}}{{Cite journal|last1=Kunin|first1=Victor|last2=Engelbrektson|first2=Anna|last3=Ochman|first3=Howard|last4=Hugenholtz|first4=Philip|date=2010|title=Wrinkles in the rare biosphere: pyrosequencing errors can lead to artificial inflation of diversity estimates|journal=Environmental Microbiology|volume=12|issue=1|pages=118–123|doi=10.1111/j.1462-2920.2009.02051.x|pmid=19725865|s2cid=19870165 |issn=1462-2920|url=https://digital.library.unt.edu/ark:/67531/metadc932456/}} 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.{{Cite journal|last1=Rob Knight|last2=Reeder|first2=Jens|date=2009|title=The 'rare biosphere': a reality check|journal=Nature Methods|volume=6|issue=9|pages=636–637|doi=10.1038/nmeth0909-636|pmid=19718016|s2cid=5278501|issn=1548-7105}} However, real rare taxa may exist among these low-abundance reads.{{Cite journal|last1=Zhan|first1=Aibin|last2=Hulák|first2=Martin|last3=Sylvester|first3=Francisco|last4=Huang|first4=Xiaoting|last5=Adebayo|first5=Abisola A.|last6=Abbott|first6=Cathryn L.|last7=Adamowicz|first7=Sarah J.|last8=Heath|first8=Daniel D.|last9=Cristescu|first9=Melania E.|date=2013|title=High sensitivity of 454 pyrosequencing for detection of rare species in aquatic communities|journal=Methods in Ecology and Evolution|volume=4|issue=6|pages=558–565|doi=10.1111/2041-210X.12037|hdl=11336/2674 |s2cid=53576369 |issn=2041-210X|doi-access=free|hdl-access=free}} 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.{{Cite journal|last1=Zhan|first1=Aibin|last2=He|first2=Song|last3=Brown|first3=Emily A.|last4=Chain|first4=Frédéric J. J.|last5=Therriault|first5=Thomas W.|last6=Abbott|first6=Cathryn L.|last7=Heath|first7=Daniel D.|last8=Cristescu|first8=Melania E.|last9=MacIsaac|first9=Hugh J.|date=2014|title=Reproducibility of pyrosequencing data for biodiversity assessment in complex communities|journal=Methods in Ecology and Evolution|volume=5|issue=9|pages=881–890|doi=10.1111/2041-210X.12230|issn=2041-210X|doi-access=free}} Cryptic diversity can also result in inflated biodiversity as one morphological species may actually split into many distinct molecular sequences. This will go a long way in generating DNA reference data which is crucial for environmental DNA-based biodiversity monitoring.

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.{{cite journal |last1=Chua |first1=Physilia Y. S. |last2=Bourlat |first2=Sarah J. |last3=Ferguson |first3=Cameron |last4=Korlevic |first4=Petra |last5=Zhao |first5=Leia |last6=Ekrem |first6=Torbjørn |last7=Meier |first7=Rudolf |last8=Lawniczak |first8=Mara K. N. |title=Future of DNA-based insect monitoring |journal=Trends in Genetics |date=10 March 2023 |volume=39 |issue=7 |pages=531–544 |doi=10.1016/j.tig.2023.02.012 |pmid=36907721 |s2cid=257470926 |url=https://www.sciencedirect.com/science/article/pii/S0168952523000380}}{{cite journal |last1=Srivathsan |first1=Amrita |last2=Hartop |first2=Emily |last3=Puniamoorthy |first3=Jayanthi |last4=Lee |first4=Wan Ting |last5=Kutty |first5=Sujatha Narayanan |last6=Kurina |first6=Olavi |last7=Meier |first7=Rudolf |title=Rapid, large-scale species discovery in hyperdiverse taxa using 1D MinION sequencing |journal=BMC Biology |date=December 2019 |volume=17 |issue=1 |pages=96 |doi=10.1186/s12915-019-0706-9|pmid=31783752 |pmc=6884855 |doi-access=free }}{{cite journal |last1=Srivathsan |first1=Amrita |last2=Lee |first2=Leshon |last3=Katoh |first3=Kazutaka |last4=Hartop |first4=Emily |last5=Kutty |first5=Sujatha Narayanan |last6=Wong |first6=Johnathan |last7=Yeo |first7=Darren |last8=Meier |first8=Rudolf |title=ONTbarcoder and MinION barcodes aid biodiversity discovery and identification by everyone, for everyone |journal=BMC Biology |date=December 2021 |volume=19 |issue=1 |pages=217 |doi=10.1186/s12915-021-01141-x|pmid=34587965 |pmc=8479912 |doi-access=free }}{{cite journal |last1=Srivathsan |first1=Amrita |last2=Baloğlu |first2=Bilgenur |last3=Wang |first3=Wendy |last4=Tan |first4=Wei X. |last5=Bertrand |first5=Denis |last6=Ng |first6=Amanda H. Q. |last7=Boey |first7=Esther J. H. |last8=Koh |first8=Jayce J. Y. |last9=Nagarajan |first9=Niranjan |last10=Meier |first10=Rudolf |title=A MinION™-based pipeline for fast and cost-effective DNA barcoding |journal=Molecular Ecology Resources |date=September 2018 |volume=18 |issue=5 |pages=1035–1049 |doi=10.1111/1755-0998.12890|pmid=29673082 |s2cid=4982474 }}{{cite journal |last1=Meier |first1=Rudolf |last2=Wong |first2=Winghing |last3=Srivathsan |first3=Amrita |last4=Foo |first4=Maosheng |title=$1 DNA barcodes for reconstructing complex phenomes and finding rare species in specimen-rich samples |journal=Cladistics |date=February 2016 |volume=32 |issue=1 |pages=100–110 |doi=10.1111/cla.12115|pmid=34732017 |s2cid=83862072 |doi-access=free }}File:Megabarcoding.pngThis 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.{{cite journal |last1=Hebert |first1=Paul D. N. |last2=Braukmann |first2=Thomas W. A. |last3=Prosser |first3=Sean W. J. |last4=Ratnasingham |first4=Sujeevan |last5=deWaard |first5=Jeremy R. |last6=Ivanova |first6=Natalia V. |last7=Janzen |first7=Daniel H. |last8=Hallwachs |first8=Winnie |last9=Naik |first9=Suresh |last10=Sones |first10=Jayme E. |last11=Zakharov |first11=Evgeny V. |title=A Sequel to Sanger: amplicon sequencing that scales |journal=BMC Genomics |date=27 March 2018 |volume=19 |issue=1 |pages=219 |doi=10.1186/s12864-018-4611-3|pmid=29580219 |pmc=5870082 |doi-access=free }}

Megabarcoding can help fill the dark taxa. DNA barcode reference data gap for insects and accelerate species discovery,{{cite journal |last1=Srivathsan |first1=Amrita |last2=Ang |first2=Yuchen |last3=Heraty |first3=John M. |last4=Hwang |first4=Wei Song |last5=Jusoh |first5=Wan F.A. |last6=Kutty |first6=Sujatha Narayanan |last7=Puniamoorthy |first7=Jayanthi |last8=Yeo |first8=Darren |last9=Roslin |first9=Tomas |last10=Meier |first10=Rudolf |title=Global convergence of dominance and neglect in flying insect diversity |date=4 August 2022 |doi=10.1101/2022.08.02.502512|s2cid=251369606 |url=https://www.biorxiv.org/content/10.1101/2022.08.02.502512v1 |website=bioRxiv}}{{cite journal |last1=Fernandez-Triana |first1=Jose L. |title=Turbo taxonomy approaches: lessons from the past and recommendations for the future based on the experience with Braconidae (Hymenoptera) parasitoid wasps |journal=ZooKeys |date=25 February 2022 |issue=1087 |pages=199–220 |doi=10.3897/zookeys.1087.76720|pmid=35585942 |pmc=8897373 |doi-access=free }} understand species diversity patterns,{{cite journal |last1=Baloğlu |first1=Bilgenur |last2=Clews |first2=Esther |last3=Meier |first3=Rudolf |title=NGS barcoding reveals high resistance of a hyperdiverse chironomid (Diptera) swamp fauna against invasion from adjacent freshwater reservoirs |journal=Frontiers in Zoology |date=December 2018 |volume=15 |issue=1 |pages=31 |doi=10.1186/s12983-018-0276-7|pmid=30127839 |pmc=6092845 |doi-access=free }}{{cite journal |last1=Yeo |first1=Darren |last2=Srivathsan |first2=Amrita |last3=Puniamoorthy |first3=Jayanthi |last4=Maosheng |first4=Foo |last5=Grootaert |first5=Patrick |last6=Chan |first6=Lena |last7=Guénard |first7=Benoit |last8=Damken |first8=Claas |last9=Wahab |first9=Rodzay A. |last10=Yuchen |first10=Ang |last11=Meier |first11=Rudolf |title=Mangroves are an overlooked hotspot of insect diversity despite low plant diversity |journal=BMC Biology |date=14 September 2021 |volume=19 |issue=1 |pages=202 |doi=10.1186/s12915-021-01088-z|pmid=34521395 |pmc=8442405 |doi-access=free }}{{cite journal |last1=Geiger |first1=Matthias |last2=Moriniere |first2=Jerome |last3=Hausmann |first3=Axel |last4=Haszprunar |first4=Gerhard |last5=Wägele |first5=Wolfgang |last6=Hebert |first6=Paul |last7=Rulik |first7=Björn |title=Testing the Global Malaise Trap Program – How well does the current barcode reference library identify flying insects in Germany? |journal=Biodiversity Data Journal |date=1 December 2016 |volume=4 |issue=4 |pages=e10671 |doi=10.3897/BDJ.4.e10671|pmid=27932930 |pmc=5136679 |doi-access=free }} evaluate species richness,{{cite journal |last1=Hebert |first1=Paul D. N. |last2=Ratnasingham |first2=Sujeevan |last3=Zakharov |first3=Evgeny V. |last4=Telfer |first4=Angela C. |last5=Levesque-Beaudin |first5=Valerie |last6=Milton |first6=Megan A. |last7=Pedersen |first7=Stephanie |last8=Jannetta |first8=Paul |last9=deWaard |first9=Jeremy R. |title=Counting animal species with DNA barcodes: Canadian insects |journal=Philosophical Transactions of the Royal Society B: Biological Sciences |date=5 September 2016 |volume=371 |issue=1702 |pages=20150333 |doi=10.1098/rstb.2015.0333|pmid=27481785 |pmc=4971185 }} generate rapid biodiversity species inventories,{{cite journal |last1=Telfer |first1=Angela |last2=Young |first2=Monica |last3=Quinn |first3=Jenna |last4=Perez |first4=Kate |last5=Sobel |first5=Crystal |last6=Sones |first6=Jayme |last7=Levesque-Beaudin |first7=Valerie |last8=Derbyshire |first8=Rachael |last9=Fernandez-Triana |first9=Jose |last10=Rougerie |first10=Rodolphe |last11=Thevanayagam |first11=Abinah |last12=Boskovic |first12=Adrian |last13=Borisenko |first13=Alex |last14=Cadel |first14=Alex |last15=Brown |first15=Allison |last16=Pages |first16=Anais |last17=Castillo |first17=Anibal |last18=Nicolai |first18=Annegret |last19=Glenn Mockford |first19=Barb Mockford |last20=Bukowski |first20=Belén |last21=Wilson |first21=Bill |last22=Trojahn |first22=Brock |last23=Lacroix |first23=Carole Ann |last24=Brimblecombe |first24=Chris |last25=Hay |first25=Christoper |last26=Ho |first26=Christmas |last27=Steinke |first27=Claudia |last28=Warne |first28=Connor |last29=Garrido Cortes |first29=Cristina |last30=Engelking |first30=Daniel |last31=Wright |first31=Danielle |last32=Lijtmaer |first32=Dario |last33=Gascoigne |first33=David |last34=Hernandez Martich |first34=David |last35=Morningstar |first35=Derek |last36=Neumann |first36=Dirk |last37=Steinke |first37=Dirk |last38=Marco DeBruin |first38=Donna DeBruin |last39=Dobias |first39=Dylan |last40=Sears |first40=Elizabeth |last41=Richard |first41=Ellen |last42=Damstra |first42=Emily |last43=Zakharov |first43=Evgeny |last44=Laberge |first44=Frederic |last45=Collins |first45=Gemma |last46=Blagoev |first46=Gergin |last47=Grainge |first47=Gerrie |last48=Ansell |first48=Graham |last49=Meredith |first49=Greg |last50=Hogg |first50=Ian |last51=McKeown |first51=Jaclyn |last52=Topan |first52=Janet |last53=Bracey |first53=Jason |last54=Guenther |first54=Jerry |last55=Sills-Gilligan |first55=Jesse |last56=Addesi |first56=Joseph |last57=Persi |first57=Joshua |last58=Layton |first58=Kara |last59=D'Souza |first59=Kareina |last60=Dorji |first60=Kencho |last61=Grundy |first61=Kevin |last62=Nghidinwa |first62=Kirsti |last63=Ronnenberg |first63=Kylee |last64=Lee |first64=Kyung Min |last65=Xie |first65=Linxi |last66=Lu |first66=Liuqiong |last67=Penev |first67=Lyubomir |last68=Gonzalez |first68=Mailyn |last69=Rosati |first69=Margaret |last70=Kekkonen |first70=Mari |last71=Kuzmina |first71=Maria |last72=Iskandar |first72=Marianne |last73=Mutanen |first73=Marko |last74=Fatahi |first74=Maryam |last75=Pentinsaari |first75=Mikko |last76=Bauman |first76=Miriam |last77=Nikolova |first77=Nadya |last78=Ivanova |first78=Natalia |last79=Jones |first79=Nathaniel |last80=Weerasuriya |first80=Nimalka |last81=Monkhouse |first81=Norman |last82=Lavinia |first82=Pablo |last83=Jannetta |first83=Paul |last84=Hanisch |first84=Priscila |last85=McMullin |first85=R. 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File:DNA_(meta)barcoding_differences.pdf

{{main|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.

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.{{Cite book|title=Environmental DNA : for biodiversity research and monitoring|last=Pierre|first=Taberlet|others=Bonin, Aurelie, 1979-|isbn=9780191079993|location=Oxford|oclc=1021883023|date = 2018-02-02}} 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.

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.{{Cite journal|last1=Ruppert|first1=Krista M.|last2=Kline|first2=Richard J.|last3=Rahman|first3=Md Saydur|date=January 2019|title=Past, present, and future perspectives of environmental DNA (eDNA) metabarcoding: A systematic review in methods, monitoring, and applications of global eDNA|journal=Global Ecology and Conservation|volume=17|pages=e00547|doi=10.1016/j.gecco.2019.e00547|doi-access=free}}{{Cite journal|last1=Stoeck|first1=Thorsten|last2=Frühe|first2=Larissa|last3=Forster|first3=Dominik|last4=Cordier|first4=Tristan|last5=Martins|first5=Catarina I.M.|last6=Pawlowski|first6=Jan|date=February 2018|title=Environmental DNA metabarcoding of benthic bacterial communities indicates the benthic footprint of salmon aquaculture|journal=Marine Pollution Bulletin|volume=127|pages=139–149|doi=10.1016/j.marpolbul.2017.11.065|pmid=29475645|doi-access=free}}

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

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.{{Cite journal|last1=Evans|first1=Darren M.|last2=Kitson|first2=James J. N.|last3=Lunt|first3=David H.|last4=Straw|first4=Nigel A.|last5=Pocock|first5=Michael J. O.|date=2016|title=Merging DNA metabarcoding and ecological network analysis to understand and build resilient terrestrial ecosystems|journal=Functional Ecology|volume=30|issue=12|pages=1904–1916|doi=10.1111/1365-2435.12659|issn=1365-2435|url=http://nora.nerc.ac.uk/id/eprint/514416/1/N514416PP.pdf}} However, there are current joined attempts, like e.g. the EU COST network [https://dnaqua.net/ DNAqua-Net], to move forward by exchanging experience and knowledge to establish best-practice standards for biomonitoring.

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.{{cite journal |title=Magnetically Recoverable, Thermostable, Hydrophobic DNA/Silica Encapsulates and Their Application as Invisible Oil Tags|author1=Puddu, M. |author2=Paunescu, D. |author3=Stark, W.J. |author4=Grass, R.N. |year=2014|doi=10.1021/nn4063853 |pmid=24568212 |volume=8 |issue = 3|journal=ACS Nano |pages=2677–2685}}

Subtopics:

{{div col|content=

• Algae DNA barcoding
• Microbial DNA barcoding
• Pollen DNA barcoding
• Aquatic macroinvertebrate DNA barcoding
• Fungal DNA barcoding
• Fish DNA barcoding
• Metabarcoding
• DNA barcoding in diet assessment

}}

Related topics:

{{div col|content=

• DNA profiling
• Consortium for the Barcode of Life

}}

Also see the sidebar navigation at the top of the article.

{{Reflist}}

• [http://swebol.org/ SweBOL]
• [http://www.finbol.org/ FinBOL]
• [http://ibol.org/ International Barcode of Life Project (iBOL)]
• [http://v4.boldsystems.org/index.php/Public_BINSearch?searchtype=records BOLD]
• [https://unite.ut.ee/ UNITE]
• [https://www6.inra.fr/carrtel-collection/Barcoding-database Diat.barcode]{{Dead link|date=January 2024 |bot=InternetArchiveBot |fix-attempted=yes }}

{{Phylogenetics}}

Category:DNA barcoding