DNA barcoding

DNA barcoding is a method of species

identification using a short section of
DNA from a specific gene or genes. The
premise of DNA barcoding is that, by
comparison with a reference library of
such DNA sections (also called
"sequences"), an individual sequence can
be used to uniquely identify an organism
DNA barcoding scheme
to species, in the same way that 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, parts of an organism, or simply to catalog as many taxa as possible, or to
compare with traditional taxonomy in an effort to determine species boundaries.

Different gene regions are used to identify the different organismal groups using barcoding. The most
commonly used barcode region for animals and some protists is a portion of the cytochrome c oxidase I (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.[2][3] 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".[4]

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.[5] When barcoding is used to identify organisms from a sample containing DNA from
more than one organism, the term DNA metabarcoding is used,[6][7] e.g. DNA metabarcoding of diatom
communities in rivers and streams, which is used to assess water quality.[8]

Contents
Background
Methodology
Sampling and preservation
DNA extraction, amplification and sequencing
Marker selection
Reference libraries and bioinformatics
Bioinformatic analysis
Species identification and taxonomic assignment
Applications
Identification of species
Detection of invasive species
 Delimiting cryptic species
Diet analysis and food web application
Barcoding for food safety
Biomonitoring and ecological assessment
Potentials and shortcomings
Potentials
Time and cost
Taxonomic resolution
Shortcomings
Physical parameters
Technological bias
Lack of standardization
Mismatches between conventional (morphological) and barcode based identification
Estimates of richness/diversity
DNA metabarcoding
Methodology
Applications
Advantages and challenges
See also
References
External links

Background
DNA barcoding techniques were developed from early DNA sequencing work on microbial communities
using the 5S rRNA gene.[9] 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.[10] 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[10] as a suitable discriminatory tool between metazoan
invertebrates.[11] 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".

Methodology

Sampling and preservation

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.[12] Examples of bulk samples
include aquatic macroinvertebrate sample 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 as 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 leave 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.[13]

DNA extraction, amplification and sequencing

DNA barcoding requires that DNA in the sample is extracted. Several different DNA extraction methods
exist, and factors like cost, time, sample type and yield affect the selection of the optimal method.

When DNA from organismal or eDNA samples is amplified using polymerase chain reaction (PCR), the
reaction can be affected negatively by inhibitor molecules contained in the sample.[14] 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)[15] 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.[16][17]

When the DNA barcode marker region has been amplified, the next step is to sequence the marker region
using DNA sequencing methods.[18] Many different sequencing platforms are available, and technical
development is proceeding rapidly.

Marker selection
Markers used for DNA barcoding are called barcodes. In order to
successfully characterize species based on DNA barcodes, selection
of informative DNA regions is crucial. A good DNA barcode should
have low intra-specific and high inter-specific variability[10] 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.[19]

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,[20] or depending on the study question.
HiSeq sequencers at SciLIfeLab in
For animals, the most widely used barcode is mitochondrial Uppsala, Sweden. The photo was
cytochrome C oxidase I (COI) locus.[21] Other mitochondrial genes, taken during the excursion of SLU
such as Cytb, 12S or 18S are also used. Mitochondrial genes are course PNS0169 in March 2019.
preferred over nuclear genes because of their lack of introns, their
haploid
mode of

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.

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.[20]

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.[20] 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 (LSU).[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.[20]

Markers that have been used for DNA barcoding in different organism groups, modified
from Purty and Chatterjee.[20]
Organism group Marker gene/locus

Animals COI,[33] Cytb,[34] 12S,[35] 16S[36]

Plants matK,[37] rbcL,[38] psbA-trnH,[39] ITS[40]

Bacteria COI,[26] rpoB,[28] 16S,[41] cpn60,[27] tuf,[42] RIF,[43] gnd[44]

Fungi ITS,[2][45] TEF1α,[46][47] RPB1 (LSU), RPB2 (LSU), 18S (SSU)[32]

Protists ITS,[48] COI,[49] rbcL,[50] 18S,[51] 28S[50]

Reference libraries and bioinformatics

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.[52]
In the microbial world, there is no DNA information for most species names, and many DNA sequences
cannot be assigned to any Linnaean binomial.[53] 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).[54]

BOLD (http://v4.boldsystems.org/index.php/Public_BINSearch?searchtype=records)

Launched in 2007, the Barcode of Life Data System (BOLD)[55] is one of the biggest databases, containing
more than 450 000 BINs (Barcode Index Numbers) in 2019. 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.
UNITE (https://unite.ut.ee/)

The UNITE database[56] was launched in 2003 and is a reference database for the molecular identification of
fungal species with the 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 (https://www6.inra.fr/carrtel-collection/Barcoding-database)

Diat.barcode[57] database was first published under the name R-syst::diatom[58] 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

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,[59] Uparse,[60] Qiime,[61] Galaxy,[62] Obitools,[63] JAMP,[64]
Barque,[65] and DADA2.[66]
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.[67]

Species identification and taxonomic assignment

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.[68] 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
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,[69] linking egg and larval stages to adult species, securing intellectual property rights for bioresources,
framing global management plans for conservation strategies and elucidate feeding niches.[70] 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

Specific short DNA sequences or markers from a standardized region of the genome can provide a DNA
barcode for identifying species.[71] 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.[72] 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.[73]

Detection of invasive species

Alien species can be detected via barcoding.[74][75] 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[74] 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.[76]

Delimiting cryptic species

DNA barcoding enables the identification and recognition of cryptic species.[77] 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.[78] 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.[79] 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.[80] 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%.[81]

Diet analysis and food web

application

DNA barcoding and metabarcoding can be useful in

diet analysis studies,[82] and is typically used if prey
specimens cannot be identified based on
morphological characters.[83][84] There is a range of
sampling approaches in diet analysis: DNA
metabarcoding can be conducted on stomach
contents,[85] feces,[84][86] saliva[87] or whole body
analysis.[69][88] In fecal samples or highly digested
stomach contents, it is often not possible to Barcoding is a tool to vouch for food quality. Here, DNA
from traditional Norwegian Christmas food is extracted
distinguish tissue from single species, and therefore
at the molecular systematic lab at NTNU University
metabarcoding can be applied instead.[84][89] Feces
Museum.
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

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,[90] or to separate poisonous mushrooms from
edible ones (Ref).

Biomonitoring and ecological assessment

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.
Potentials and shortcomings

Potentials

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.[91]

Time and cost

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

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.[92] 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.[93]
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 [94] 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 (http://www.terrain.net.nz/friends-of-te-henui-group/invertebrates-freshwater-new-zealand/may
fly-nymph-genus-deleatidium.html). 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.[95]

Shortcomings
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.[91] This recommendation is based on multiple
perceived challenges.

Physical parameters

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).[96][97] 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.[98] Therefore, best practice for DNA barcoding is to
sequence vouchered specimens.[99][100] 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.[98] 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.[101]

Technological bias

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.[102][103] 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.[104] However, primers will often bind preferentially to some sequences, leading to differential primer
efficiency and specificity and unrepresentative communities’ assessment and richness inflation.[105] 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 [106][107] or PCR-free approaches to
avoid these biases, but as of today, the DNA metabarcoding technique is still based on the sequencing of
amplicons.[104] Other bias enter the picture during the sequencing and during the bioinformatic processing of
the sequences, like the creation of chimeras.

Lack of standardization
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.[104] 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 [108] and is also addressed by CEN (the European
Committee for Standardization).[109]

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 (Ref needed).

Mismatches between conventional (morphological) and barcode based identification

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.[91] 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.[91]

Estimates of richness/diversity

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.[110][111] 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.[112] However, real rare taxa may exist among these low-
abundance reads.[113] 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.[114] Cryptic diversity can also result in inflated biodiversity as one morphological
species may actually split into many distinct molecular sequences.[91]

DNA metabarcoding
DNA 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

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 Differences in the standard methods for DNA barcoding & metabarcoding. While
highly conserved gene DNA barcoding points to find a specific species, metabarcoding looks for the
regions which can be used whole community.
for primer design.[12]
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

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.[115][116]

DNA metabarcoding applications include:

Biodiversity monitoring in terrestrial and aquatic environments

Paleontology and ancient ecosystems
Plant-pollinator interactions
Diet analysis
Food safety

Advantages and challenges

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.[117] However, there are
current joined attempts, like e.g. the EU COST network DNAqua-Net (https://dnaqua.net/), to move forward
by exchanging experience and knowledge to establish best-practice standards for biomonitoring.[91]

See also
Microbial DNA barcoding
Algae DNA barcoding
Fungal DNA barcoding
 Fish DNA barcoding
Pollen DNA barcoding
DNA barcoding in diet assessment
DNA profiling
Consortium for the Barcode of Life

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(PDF). Functional Ecology. 30 (12): 1904–1916. doi:10.1111/1365-2435.12659 (https://doi.org/
10.1111%2F1365-2435.12659). ISSN 1365-2435 (https://www.worldcat.org/issn/1365-2435).

External links
SweBOL (http://swebol.org/)
FinBOL (http://www.finbol.org/)
International Barcode of Life Project (iBOL) (http://ibol.org/)
BOLD (http://v4.boldsystems.org/index.php/Public_BINSearch?searchtype=records)
UNITE (https://unite.ut.ee/)
Diat.barcode (https://www6.inra.fr/carrtel-collection/Barcoding-database)

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