DNA Barcoding, An Effective Tool For Species Identification: A Review

Molecular Biology Reports (2023) 50:761–775
https://doi.org/10.1007/s11033-022-08015-7
REVIEW
DNA barcoding, an effective tool for species identification: a review

Sandeep Antil1 · Jeeva Susan Abraham1 · S. Sripoorna1 · Swati Maurya1 · Jyoti Dagar1 · Seema Makhija1 ·
Pooja Bhagat1 · Renu Gupta2 · Utkarsh Sood3 · Rup Lal3 · Ravi Toteja1
Received: 4 May 2022 / Accepted: 7 October 2022 / Published online: 29 October 2022
© The Author(s), under exclusive licence to Springer Nature B.V. 2022
Abstract
DNA barcoding is a powerful taxonomic tool to identify and discover species. DNA barcoding utilizes one or more standard-
ized short DNA regions for taxon identification. With the emergence of new sequencing techniques, such as Next-generation
sequencing (NGS), ONT MinION nanopore sequencing, and Pac Bio sequencing, DNA barcoding has become more accu-
rate, fast, and reliable. Rapid species identification by DNA barcodes has been used in a variety of fields, including forensic
science, control of the food supply chain, and disease understanding. The Consortium for Barcode of Life (CBOL) presents
various working groups to identify the universal barcode gene, such as COI in metazoans; rbcL, matK, and ITS in plants; ITS
in fungi; 16S rRNA gene in bacteria and archaea, and creating a reference DNA barcode library. In this article, an attempt has
been made to analyze the various proposed DNA barcode for different organisms, strengths & limitations, recent advance-
ments in DNA barcoding, and methods to speed up the DNA barcode reference library construction. This study concludes
that constructing a reference library with high species coverage would be a major step toward identifying species by DNA
barcodes. This can be achieved in a short period of time by using advanced sequencing and data analysis methods.
Keywords Biodiversity · DNA barcoding · Next-generation sequencing (NGS) · Oxford Nanopore Technologies (ONT)’s
MinION™ · PacBio sequencing · Consortium for barcode of life (CBOL)
Introduction among living organisms from all sources comprising ter-

restrial, marine, and other aquatic ecosystems and ecologi-
The existence of life is one of the most unique aspects cal complexes of which they are a part; it encompasses
of Earth, and the diversity of life is the most astonishing diversity within species, between species, and within eco-
feature of life. Biological diversity refers to the variation systems [1]. Biodiversity plays a key role in maintaining
* Ravi Toteja Renu Gupta

ravitoteja@andc.du.ac.in rgupta17@maitreyi.du.ac.in
Sandeep Antil Utkarsh Sood
sandeep@andc.du.ac.in c_utkarsh.sood@teri.res.in
Jeeva Susan Abraham Rup Lal
jeevasa@andc.du.ac.in rup.lal@teri.res.in
S. Sripoorna 1
Acharya Narendra Dev College, University of Delhi,
sripoorna@andc.du.ac.in
New Delhi, Delhi, India
Swati Maurya 2
Maitreyi College, University of Delhi, New Delhi,
swatimaurya@andc.du.ac.in
Delhi 110 021, India
Jyoti Dagar 3
The Energy and Resources Institute, IHC Complex,
jyotidagar@andc.du.ac.in
New Delhi 110003, India
Seema Makhija
seemamakhija@andc.du.ac.in
Pooja Bhagat
poojabhagat@andc.du.ac.in
13
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762 Molecular Biology Reports (2023) 50:761–775
ecological balance [2]. The diversity of species described satisfy three criteria (1) A distinct ‘barcoding gap’ between
so far is a very tiny portion of biodiversity present on the maximum intra-specific and inter-specific divergence within
earth (approximately 9 million). This means that it is very a group of organisms; (2) conserved flanking sites for cre-
hard to estimate the diversity of life that go extinct every ating universal PCR primers; (3) short sequence length to
day, as scientists have only described 10–15% of the total facilitate current capabilities of DNA extraction and ampli-
diversity of the earth [3]. A considerable portion around fication [8].
86–91% (~ 7.2 million) of diversity remains undescribed due This technique involves a collection of a sample from
to several reasons such as scarcity of funds for taxonomy, a the field, extracting DNA, selection of barcoding gene for
very less number of the trained taxonomist, and absence of amplification by using a universal primer (Table 1), ampli-
accurate species identification methods. For describing the fied DNA molecule is sequenced by Sanger sequencing or
remaining diversity an accurate method for taxonomic iden- High-throughput sequencing for assessing the diversity and
tification, trained taxonomist, and funding are required. Tra- analysis of obtained data by using data analysis software
ditionally, taxonomic assessment has relied on the basis of such as Mothur, Qiime2 etc. (Fig. 1) [10, 11].
morphological character which is time-consuming, requires The Consortium for the Barcode of Life (CBOL, https://
taxonomic specialists, and gives false identification when www.ibol.org) is an international organisation that was
cryptic species and phenotypic plasticity are concerned [4, founded in 2004 to facilitate the establishment and use of
5]. Furthermore, globally, the number of traditional taxono- DNA barcodes as a global standard for biological species
mists is declining [6]. Therefore, the majority of the diver- identification. CBOL includes various group, such as Plant
sity of microorganisms and invertebrates may have to be working group for plants, Protist working group for eukar-
distinguished solely by DNA-based molecular techniques, yotic microorganism, Fungal working group for fungi, to
without accompanying live cultures or physical specimens. identify the universal barcode gene and creating a reference
These molecular techniques have several advantages over DNA barcode library [12]. A reference database, Barcode of
traditional approaches. Because, molecular tools are stand- life data system (BOLD, http://www.boldsystems.org), has
ardized tools which allow direct comparison among different been developed that aids in acquiring, storage, analysis, and
users and they do not require taxonomic expertise, can be publication of DNA barcode and allows a significant number
applied to environmental samples which comprise a mix of of species to be identified [13]. The present study aims to
several species, like soil or a water sample and can be used review the various proposed/available DNA barcodes the
in early warning allowing detection of low concentration of for animals, plants, fungi, bacteria, virus, and protists (more
potential invaders, or even imprints of potential invader [7]. specifically ciliates). Over the period of time, significant
Over the last decade, DNA barcoding emerges as a new advances have been made in DNA barcoding. One impor-
molecular tool for taxonomists to identify species. DNA tant advancement in barcoding by mitogenomics and nuclear
barcoding utilizes one or more standardized short genetic ribosomal RNA repeats obtained by genome skimming. The
markers in an organism’s DNA to recognize it as belong- same has been discussed in the present review along with the
ing to a particular species, and through this strategy, DNA mitogenomics approach for species identification. In the end,
sample from the unidentified species is compared to identi- strengths and limitations of the technique have also been
fied sequences present in a DNA barcode reference library, briefly described.
developed by Hebert and his collaborators [8]. DNA barcod-
ing is based on the principle of barcoding gap that refers to
the difference between mean intra- and interspecific genetic Barcodes for identification of animals
distances. The wider the barcoding gap is, the more reliable
species discrimination will be achieved. DNA barcoding is Hebert et al. had suggested a 650 bp fragment of the mito-
budget-friendly, less time-consuming, objective method, and chondrial cytochrome-c oxidase subunit 1 (COI) gene as a
a powerful tool for species identification when cryptic spe- universal marker or ‘DNA barcode’ for global biological
cies and phenotypic plasticity is a concern or morphology identification of animal species. COI gene is a mitochon-
keys are not available [9]. Due to the better precision and drial gene that is highly conserved [14], codes for respira-
ease of DNA barcoding, this technique is gaining popular- tory electron transport chain protein that reduce molecular
ity, and it can be used to identify species in any stage of oxygen into water, present in all aerobic organisms. Mito-
life (i.e. both adults and immature stage including eggs). chondrial genes are preferred over nuclear genes because
DNA barcoding mostly differs from other molecular tools mitochondrial genes are generally haploid, lack introns,
by use of standard markers, such as COI in metazoans; rbcL, and contain limited recombination. Mitochondria repro-
matK, and ITS in plants; ITS in fungi; 16S rRNA gene in duce by binary fission and without sexual recombination,
bacteria and archaea [7]. For DNA barcoding, the selection so the mitochondrial genes are subjected less to insertions,
of the barcoding gene is crucial. A barcoding gene must deletions or other large-scale rearrangements that introduce
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Molecular Biology Reports (2023) 50:761–775 763
Table 1 Represents list of barcoding gene for species identification and their primer pairs
Organisms Barcode gene Primers (5′–3′) References
Animals COI F-GGTCAACAAATCATAAAGATATTGG [15]

R-TAAACTTCAGGGTGACCAAAAAATCA
COII F-TCACWATACTAYTAACAGATCGCAA [95]
R-AATAGCTGTACAGTGGGT
Plant COI F-CAACATTT ATTTTGATTTTTTGG [16]
R-TCCAATGCACTAATCTGCCATATTA
RbcL F-ATGTCACCACAAACAGAAAC [19]
R-TCGCATGTACCTGCA
MatK F-CCTCATCTGGAAATCTTGGTT [19]
R-GCTTATAATGAGAAAGATTTCTGC
PsbA-trnH F-CGCGCATGGTGGATTCACAATCC [19]
R-GTTATGCATGAACGTAATGCTC
rpoC1 F-CCSATTGTATGGGAAATACTT [96]
R-CTTACAAACTAATGGATGTAA
rpoB F1-AAGTGCATTGTTGGAACTGG [19]
F2-ATGCAACGTCAAGCAGTTCC
R1-CCGTATGTGAAAAGAAGTATA
R2-GATCCCAGCATCACAATTCC
atpF-atpH F-ACTCGCACACACTCCCTTTCC [19]
R-GCTTTTATGGAAGCTTTAACAAT
psbK-psbI F-TTAGCCTTTGTTTGGCAAG [19]
R-AGAGTTTGAGAGTAAGCAT
ITS F-ATGCGATACTTGGTGTGAAT [9]
R-GACGCTTCTCCAGACTACAAT
Fungi ITS F-TCCTCCGCTTATTGATATGC [21, 24]
R-GGAAGTAAAAGTCGTAACAAGG
D1-D2 F-ACCCGCTGAACTTAAGC [22]
R-TCCTGAGGGAAACTTCG
RPB1 F-GARTGYCCDGGDCAYTTYGG [24]
R-CCNGCDATNTCRTTRTCCATRTA
RPB2 F-GAYGAYMGWGATCAYTTYGG
R-CCCATWGCYTGCTTMCCCAT
COI F1-TTACAAGGTGATCATCAATT [23]
F2-GTATTAAAATTTCTATCTGTAAG
R1-TTTCTATCTGTAAGTAACAT
R2-TTTACAAGGTGATCAA
ACT F-ATGTGCAAGGCCGGTTTCG [97]
R-TACGAGTCCTTCTGGCCCAT
TEF1a F-GCYCCYGGHCAYCGTTTYAT [98]
R-ACHGTRCCRATACCACCRATCTT
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Table 1 (continued)
Organisms Barcode gene Primers (5′–3′) References
Protist ITS F-CGTAACAAGGTTTCCGTAGG [45]

R-TCCTCCGCTTACTGATATGC
V4 F-CCAGCASCYGCGGTAATTCC [45]
R-ACTTTCGTTCTTGATYRA
V9 F-GTACACACCGCCCGTC [42]
R-TGATCCTTCTGCAGGTTCACCTAC
D1-D2/D2-D3 F-AGCGGAGGAAAAGAAACTA [42]
R-ACGATCGATTTGCACGTCAG
D3-D5 F-GACCCGTCTTGAAACACGGA [50]
R-TCGGAAGGAACCAGCTACTA
COI F-GWTGRGCKATGATYACACC [5]
R-ACCATRTACATATGATGWCC
RbcL F-TTAACCTCCATCGTGGGTAACG [99]
R-CAGGCATAGAAGCCCAATCTTG
H4 F-GGTATTACTAAGCCCGCTATCAGAAGA [53]
R-GGTCTTTCTTCTGGCGTGTTCAGTGTA
Spliced F-GTATAAGAGACAGNNNNNNN [44]
leader RNA gene R-TCAGTTTCTGTA
Archaea 16S F-GCY TAA AGS RIC CGT AGC [26]
R-TTM GGG GCA TRC IKA CCT
Type II chaperonin (High F-GGC CCG AAG GGC ATG GAC AAG ATG [26]
GC primers) R-GGC ATG TCG TCG ATG CCC TTC TG
Type II chaperonin F-GGI CCI MRR GGI ITI GAY AAR ATG [26]
(Universal primers) R-GCI AII TCR TCI ATI CCY TTY TG
Bacteria RpoB F-TTTCCCTACACGACGCTCTTCCGATCTGGYTWYGAAGTNCGH- [30]
GACGTDCA
R-GGAGTTCAGACGTGTGCTCTTCCGATCTTGACGYTGCATGT TBGM-
RCCCATMA
16S rRNA gene F-GAGTTTGATCCTGGCTCAG [29]
R-GTATTACCGCGGCTGCTG
cpn60 F1-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGAIIIIGCIGGI- [33]
GAYGGIACIACIAC
F2-GCTTCGTGGGCTCGGAGATGTGTA TAAGAGACAGYKIYKITCIC-
CRAAICC IGGIGCYTT
R1-TCGTCGGCAGCGTCAGATGTG TATAAGAGACAGGAIIIIGCIGGYGAC
GGYACSACSAC
R2-GCTTCGTGGGCTCGGAGATGTGTA TAAGAGACAGCGRCGRTCRCC
GAAGCCSGGIGCCTT
Tuf F-GCTCCTGAAGAAARAGAACGTGG [31]
R-ACTTGDCCTCTTTCKACTCTACCAGT
RIF F-TACGGCTTCGACACCTTCG [32]
R-CGGTGATCTTCTTGTTGGCG
Gnd F-CGCGGATCCGGWCCWWSWATWATGCCWGGWGG [31]
R-CGCGGGCCCGTATGWGCWCCAAAATAATCWCKTTGWGCTTG
The lower-case letters indicate nucleotides added to the genomic sequence to facilitate PCR; D = A/G/T, Y = C/T, R = A/G, W = A/T, M = A/C,
B = C/G/T, V = A/C/G, H = A/C/T, K = G/T, N = A/G/C/T
more ambiguous variation in the sequence. The mitochon- barcoding gene for moths, butterflies, collembolans, beetles,
drial genome evolves at a higher rate than the nuclear bats, spiders, wasps, ants, fishes, Reptilia, birds, chickens,
genome. Therefore, mitochondrial genomic sequences are musk deer, fruit fly, and crustacean larvae [4, 16]. Some pri-
more informative in differentiating or distinguishing closely mate taxonomists recommend that ND5 (Mitochondrial gene
related species [15]. So far COI gene has been used as encoding NADH:Ubiquinone Oxidoreductase Core Subunit
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species [19]. Comparatively, 600–800 base-pair region of

matK in association with rbcL gives the most satisfactory
result and designated as core barcoding gene, while psbA-
trnH work as a good marker for other plant species and iden-
tified as an important supplementary marker, but there is no
single marker for identifying all the plant species [20].
Barcodes for identification of fungi
Identification of fungi through morphological methods is

often difficult because they only occasionally display mor-
phological characters suitable for identification. A molecu-
lar tool such as DNA barcoding is the best way to evaluate
fungal diversity. The ITS, D1-D2 region of the large subu-
nit of ribosomal RNA gene, RPB1 and RPB2 of the large
subunit of RNA polymerase II, γ-actin (ACT), β -tubulin
II (TUB2), translation elongation factor 1-α (TEF/α), DNA
topoisomerase I (TOPI), phosphoglycerate kinase (PKG) are
used as a barcode for identifying fungal species [20–22].
COI has a higher resolution in few groups of related species
such as Penicillium, and Entolomasarcopum but in other
groups it may not give satisfactory results [23]. Schoch
and his group has proposed ITS as a universal barcode for
the identification of fungi [24]. The length of ITS region
in fungi is around 600 bp long, with two variable spacers,
ITS-1 and ITS-2, interrupted by the highly conserved 5.8S
Fig. 1 Schematic representation of the methodology for DNA barcod-
ing rRNA gene. Another significant benefit of utilizing ITS as
a barcode is that each haploid genome often contains sev-
eral tandemly repeated copies of the ribosomal rRNA gene
5) and COII should be used as a barcode in primates species cluster (including ITS), allowing it to be amplified even from
delineation and suggest that these two genes should be more small amounts of biological materials [6]. Stielow et al. have
appropriate markers than COI due to a more pronounced assessed the potentiality of D1-D2 region of LSU, β-tubulin
barcoding gap [17]. II (TUB2), γ-actin (ACT), translation elongation factor 1-α
(TEF/α), the second largest subunit of RNA-polymerase II
(RPB2), DNA topoisomerase I (TOPI), phosphoglycerate
Barcodes for identification of plants kinase (PKG), hypothetical protein LNS2 as an alternative
DNA barcode. Among these genes TEF/α has the potential
For plant species identification, the selection of barcod- as a secondary DNA barcode due to sufficient intra- and
ing genes remains very controversial. Plant mitochondrial inter-specific variation, while TOPI and PKG show high
genome exhibits a low rate of mutation (nucleotide substitu- resolution for the phylum Ascomycota, and TOPI and LNS2
tion) that restricts COI as a universal plant barcode. Plant for the subphylum Pucciniomycotina [22].
taxonomists have spent a large amount of time and found
the Chloroplast genome as an alternative to the mitochon-
drial genome. In 2009, CBOL plant working group pro- Barcodes for identification of archaea
posed seven potential barcodes such as rbcL (large subunit
of ribulose 1,5 bisphosphate carboxylase), matK (maturase Archaea is a major component of microbial diversity and
K), psbA-trnH (intergenic spacer region, rpoC1 (RNA poly- has a prominent place in the Tree of Life [25]. 16S rRNA
merase C1), rpoB (RNA polymerase B), atpF-atpH (encodes gene has been widely utilised as a barcode for evaluating
for ATP synthase subunits CFO I and CFO III) and psbK- the diversity of archaea [25, 26]. The 16S rRNA gene is not
psbI (encodes for polypeptide K and L of photosystem II) sensitive enough to discriminate closely related microbes,
[18]. Nuclear gene ITS (internal transcribed spacer) and all particularly at the species level [27]. In a study, type 2 chap-
the chloroplast barcodes have been positively tested in plant eronin or thermosome (e.g. TCP-1 ring complex/chaperonin
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containing TCP-1), which are present in both archaea and identification [12]. The hypervariable V4 region of 18S
eukaryotic cytoplasm, proposed as a potential complemen- rRNA gene is proposed as the universal eukaryotic pre-
tary barcode for 16S rRNA gene to assess the archaeal diver- barcode, while group-specific barcode is defined separately
sity since it has larger barcoding gap and generate more for each significant protistan lineage [34]. So far, ITS,
OTUs (operational taxonomic units) than 16S rRNA gene COI, rbcL,18S rRNA gene, 28S rRNA gene region have
[28]. been proposed as a protistan DNA barcode [35–37]. ITS,
the universal barcode in fungi, also has high discriminatory
power for ciliates, dinoflagellates, and oomycetes [20, 37,
Barcodes for identification of bacteria 38]. Mitochondrial COI, which is the universal barcode for
animals and default barcode for other organisms as well, is
16S rRNA gene is a universal marker as it is highly con- also positively tested in protist [36]. Hypervariable regions
served in all the species of bacteria. The length of the 16S V4 and V9 of 18S ribosomal RNA gene are promising bar-
rRNA gene is 1600 base pairs and contains nine hypervari- codes to access the diversity and phylogenetic relationship
able regions of V1–V9. More conservative regions are valu- of diatoms, dinoflagellates and ciliates [39–41]. D1-D2 and/
able for identifying higher-ranking taxa, whilst more rapidly or D2-D3 regions at 5′ end of large subunit of rRNA gene
evolving ones can aid in genus or species identification. The serve as potential barcodes for many protists lineages such
V2–V3 region of 16S rRNA gene has higher resolution for as diatoms, ciliates, and dinoflagellates [35, 42, 43]. Some
identifying lower-ranked taxa (species and genus) [29]. The group-specific barcodes such as rbcL and spliced leader
diversity of bacteria can also be accessed by using COI, RNA gene are also utilized in photosynthetic protists and
rpoB, cpn60 (encodes for chaperonin protein), tuf (elon- trypanosomatids, respectively [44].
gation factor), RIF (Replication initiation factor), and gnd
(Gluconate-6-phosphate dehydrogenase) gene as barcode
[30–33]. These genes have several benefits over frequently Barcodes for identification of ciliates
used 16S rRNA gene i.e. as they are frequently found in sin-
gle copies in bacterial genome, and develop silent mutations Large public reference libraries of DNA barcodes are being
owing to codon degeneracy, resulting in improved species developed for animals, plants, and fungi, but no universal
resolution. Of these cpn60 gives better results and can be barcode has been accepted for ciliates species identification
used as a possible alternative for assessing bacterial diversity [37]. Various barcodes for ciliate identification are (1) mito-
[20, 26] and cpn60 is the only target that can be addressed chondrial cytochrome c oxidase subunit I gene (COI gene);
with ‘universal’ PCR primers, and a curated sequence data- (2) hypervariable regions of the small subunit (SSU) rRNA
base, cpnDB, is available. For closely related species, the gene such as V4 and V9 region; (3) ITS region; (4) D1-D2
cpn60 gene has stronger discriminating power than the 16S regions of the large subunit of rRNA gene (LSU) and (5)
rRNA gene, and the uniform size and sequence variability of histone H4.
the cpn60 ‘universal target’ (UT) make sequence compari-
sons and other bioinformatics tasks easier [26].
Mitochondrial cytochrome‑c oxidase
subunit 1 gene (COI)
Barcodes for identification of viruses
Within ciliates, taxonomic and molecular phylogenetic
Viruses are the most abundant (approx. 10–12 times higher studies using COI gene have been used in Paramecium, Tet-
than the total no. of cells) life forms on earth. So far, there rahymena, Carchesium, Miamiensis, Sterkiella and Pseu-
is no standardized barcode fragment for detection of viruses dokeronopsis [5, 45]. All the above studies prove that the
[20]. highly variable COI gene of ciliates can identify closely
related species and cryptic species since it has a distinct
barcode gap between maximum intraspecific and minimum
Barcodes for identification of protist inter-specific genetic divergence (Table 1). Within ciliates,
the COI gene have been successfully sequenced from Tet-
CBOL has initiated a Protist working group (ProWG) to rahymena and Paramecium[4, 45]. The COI gene (average
identify barcode region across all protist lineages and set- 2000–2200 nucleotides long) have been found to be widely
ting up a reference DNA barcode library. CBOL ProWG has dissimilar from other eukaryotes as it includes > 300 nucleo-
introduced a 2-step pipeline for protists: first, the universal tides long insert region which has exceptional variation in a
pre-barcode to be used for preliminary identification; sec- genetic distance value and intraspecific genetic divergence
ond, a group-specific barcode to be applied for species-level [46]. This insert region is used as a barcode to discriminate
13
closely related species based on genetic divergence [8]. Ear- the most effective for revealing interspecific relationship. On
lier studies have shown that the COI gene of ciliates has high the other hand, the V9 region seems appropriate at the fam-
intraspecific genetic divergence than nuclear gene [5, 46]. ily level or higher [47]. It is recommended to use V4 and V9
Park et al. (2019) have reported a 478 bp long COI sequence together to assess the diversity and phylogenetic relationship
of 69 population of spirotricheans ciliates, which has maxi- of eukaryotic microbes [39].
mal intraspecific genetic divergence ranging from 0 to 14.8%
and minimal interspecific genetic variation, i.e.,13.6–47.3%. Internal transcribed spacer (ITS) region
They identified three putative cryptic species, Caudiholos-
tichaylvatica, Diophrys scutum, and Euplotes vannus [5]. Internal transcribed spacer (ITS) and the external transcribed
COI nucleotide tree has a higher resolution to discriminate region (ETR) are the flanking regions of the SSU, and the
closely related and sibling species at and below the spe- 5.8S rRNA is a non-coding part of LSU rRNA. ITS1 is
cies level. Recently, Zhang et al. [36] studied the phyloge- present between SSU rRNA and 5.8S rRNA, and ITS2 is
netic relationship of subclass scuticociliates with the usage present between 5.8S rRNA and LSU rRNA [45]. Various
of nuclear SSU-rRNA gene, mitochondrial SSU-rRNA studies suggest that ITS region has the potential of prom-
gene and COI gene as a molecular marker and showed that ising barcode for ciliate identification and investigation of
sequence divergence of COI (average 24%) is more signifi- intraspecific genetic diversity at species and population
cant than mtdSSU-rRNA gene (average 21%) and nSSU- levels since they shows much higher rate of evolutionary
rRNA gene (average 11.5%). They proved that COI is a bet- changes (> 100 times) than the coding regions of the ribo-
ter choice as a molecular marker to examine phylogenetic somal subunit [34, 48, 49]. Usually, phylogenetic trees of
relationships than mtdSSU-rRNA gene and nSSU-rRNA ITS1-5.8S-ITS2 region usually do not differ significantly
gene[36]. However, consortium for the barcode of life does from those inferred from the 18S rRNA gene, implying that
not consider COI as an appropriate barcode for uncovering the ITS region is a viable proxy for genealogical studies.
ciliates species because of issues like the absence of func- Although both the ITS1 and ITS2 have sufficient conserved
tional mitochondria in some ciliates from the anoxic envi- and variable region, but ITS2 seems to have more informa-
ronment e.g., ciliates belonging to Metopusand Trimyema tion and may be more valuable for comparisons at the family,
genus and presence of heteroplasmy [4, 5]. order, and even higher level. Moreover, the secondary struc-
ture of the ITS2 molecule has been employed to improve the
quality of species-level phylogenetic reconstructions. Apart
Small subunit (SSU) rRNA gene from phylogenetic reconstructions, the compensatory base
changes (CBCs) in the ITS2 region correlate with sexual
SSU rRNA gene was the first and widely used molecular incompatibility and so can be used for species discrimination
marker in genealogy and systematics study of ciliates because [48]. More and more studies suggest that using both primary
it can be sequenced accurately, universally, availability of sequence and secondary structure of ITS2 produce higher
diverse and large database from NCBI, and includes both con- phylogenetic resolution [34, 49]. Zhan et al. used ITS1-5.8S-
served and variable nucleotide sequences allowing combined ITS2 and the ITS2 as a barcode to delimitates Pseudokero-
phylogenetic reconstruction and biota recognition at various nopsis species and found that both the ITS1-5.8S-ITS2 and
taxonomic levels. Within ciliates, the average size of 18S the ITS2 regions shows similar levels of genetic variation
rRNA gene is ~ 1771 bp long except in litostomatea which has and substantial gaps between intraspecific and interspecific
1635-1641 bp [45], but this entire region of 18S rRNA gene distance (0.52–3.72% for ITS2; 0.42–3.84% for ITS-5.8S-
is not used for species identification. Only the hypervariable ITS2). Additionally, they also proposed a genetic divergence
regions (V1–V5 and V7–V9) of 18S rRNA gene are used for of 1.5% as an ideal threshold of ITS1-5.8S-ITS2 and ITS2
species identification. Among them, V4 and V9 hypervariable to distinguish Pseudokeronopsis species and also suggested
regions are considered the famous barcoding gene. The hyper- the ITS1-5.8S-ITS2 can be used as an ideal SGS (Second
variable region V9 is immensely used as a genetic marker for generation sequencing) metabarcode for assessing ciliates
evaluating eukaryotic diversity and also a prime candidate for environmental diversity [34].
assessing protist lineage richness, while the V4 region of SSU
rRNA gene is the primary candidate for studying the phyloge-
netic relationship of eukaryotes. V4 region is more extensive, Large subunit (LSU) of rRNA gene
more variable, and show better resolution to explore the evo-
lutionary relationship of eukaryotes than the V9 region [39]. LSU rRNA gene is a good barcoding gene for discriminat-
The secondary structure of hypervariable region V9, V7, V4, ing closely related taxa because it has a higher evolutionary
V2 of 18SrRNA gene in urostylids shows a high degree of rate than SSU. Similar to SSU, LSU rRNA gene has variable
variability and provides further evidence that the V4 region is region such as D1-D12, of which D1-D3 region show much
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higher variation than other variables such as D4, D5, D7, diversity instead of identifying individual taxa [54]. This
D8, D12 [50]. Over the last decade, the D1-D2 region of advancement has overcome the limitation of traditional
LSU rRNA gene emerged as a promising barcode marker for DNA barcoding, such as extensive sampling efforts. Meta-
species identification up to species level. Santoferrara et al. barcoding relies on the shorter DNA fragments instead of
has proposed D1–D2 region of LSU rRNA gene with a 1% whole 658 bp fragment (standard barcodes) used in classical
threshold value (for tintinnid) as a barcoding marker for cili- DNA barcoding. Metabarcoding approaches on environmen-
ate species identification and potentiality of this marker fur- tal and faecal samples have revealed population structure in
ther assessed by Stoeck et al., Zhao et al., Forster et al. [37, a variety of species [55]. The main problem associated with
42, 51, 52]. D1–D2 region of LSU rRNA gene has several standard barcodes is length, i.e., longer than 500 bp used in
advantages over other frequently used markers such as show- the traditional approach for achieving high discriminatory
ing a clear barcoding gap, rapid evolutionary rate enough to power at the species level. Unfortunately, metabarcoding
provide higher diversity resolution than SSU and higher uni- assess the diversity up to family, order, or higher taxonomic
versality and constant threshold value than COI [51]. LSU level from environmental DNA sample [56, 57]. One of the
has less intra-clonal and intraindividual variability [45]. One most challenging aspects of metabarcoding on which their
study suggested that the D2 region is a suitable marker for accuracy depends is to find new and acceptable primer pair
discriminating all Frontonia morphospecies since it shows a and their corresponding markers. An ideal metabarcoding
clear barcoding gap with a threshold of 4.5%, while the D1 marker should have a short length (e.g., 100 bp) for easy
region alone is not ideal for determining because it shows sequencing, good conserved flanking primer binding sites
the overlap between intraspecific and interspecific genetic to minimise taxonomic bias during PCR amplification,
divergence [37]. So far, D1–D2 region of LSU together have and a sufficiently variable intervening sequence for species
been used as a marker for diatoms, dinoflagellates, tintinnid identification [58]. V4 region is the primary choice meta-
ciliates, Paramecium and Frontonia species [37, 42, 51, 52]. barcode for assessing the richness and phylogenetic rela-
All the above-discussed features such as higher universality, tionship of eukaryotic microorganisms, while COI is widely
conserved primers for its amplification in ciliates and con- used for animals [34]. Primers with fewer template–primer
stant threshold value as well as the presence of high quality mismatches are better for quantitative DNA metabarcod-
manually curated databases (i.e., SILVA), makes hypervari- ing, especially for species of higher relative abundance in
able D1–D2 region of LSU rRNA gene promising DNA bar- a sample. Barcode of life DATABase (BOLD) system has
codes for ciliates species delineation [37]. a primer database (http://boldsystem.org/index.php/Public_
Primer_PrimerSearch) that store all the published primers.
Researchers can either determine the primers of their interest
Histone H4 by searching in primer database or design their primer by
using software like Primer3, QPRIMER, UniPrime, Prima-
The histone H4 is known to be a highly conserved protein clade, Amplicon program, Primer Hunter, Greene SCPrimer
among all eukaryotes with the exception of the high degree andecoPrimer etc.
of variation observed in the ciliate species [45]. The his- Next-generation sequencing (NGS) is a cost and time-
tone protein is responsible for the organization of eukaryotic saving high throughput platform and generate millions of
chromatin. The ciliate histone H4 encoded by the macronu- reads in a single run for only one environmental sample.
clear gene. Due to considerable difference within ciliates, Braukmann et al. compare the performance of three Next-
histone H4 is considered an excellent molecular marker to generation platforms, namely Illumina MiSeq, Ion Tor-
study phylogenetic relationships and can be used as DNA rentS5, and Ion Torrent PGM, and showed that they per-
Barcoding [53]. form equally well for species recovery, although MiSeq is
often recommended because of its low error rate and well-
established bioinformatics methods [59]. Illumina NovaSeq
Advancement in DNA barcoding is the recent advancement in sequencing technology with the
same sequencing depth as MiSeq but assesses more meta-
By using DNA metabarcoding and microarray, it is very zoan diversity. One of the known limitations of NGS for
feasible to develop a powerful taxonomic identification metabarcoding is the generation of short read length, i.e.,
tool. The development of metabarcoding was compelled 400 bp [7]. The development of Illumina MiSeq overcome
by the growth of next-generation sequencing technologies this limitation of short read length by generating longer
capable of producing millions of sequences at a compara- sequence reads (600–800 bp) that provide better taxonomic
bly low price. The Metabarcoding approach uses the same resolution and phylogenetic inference [7, 54]. Metabarcod-
general principle as the traditional DNA barcoding, but ing data has significantly improved the estimates of micro-
this approach focuses on assessing the community’s whole bial communities and offered precise information about the
13
structure and spatiotemporal turnover of microbial popu- 68]. Nanopore sequencing has also proven to be a very ver-
lations, particularly in the ocean. According to some esti- satile technology, e.g., allowing for whole genome sequenc-
mates, there are 50,000 to 100,000 protist OTUs (operational ing and assembly of fungal and human genomes, as well as
taxonomic unit) in the world’s oceans, which is five to ten sequencing full-length RNA transcripts using both direct
times the number of bacteria and archaea combined. These RNA and cDNA sequencing [69].
OTUs have different distribution patterns, with varied ocean PacBio sequencing, which is a single molecule real time
regions have various ecosystems in terms of taxonomic com- sequencing, is an alternative DNA barcoding approach for
position and relative abundances. The metabarcoding data large sample sizes: its workflow simplifies and reduces post-
also used to relate microbial community distribution patterns sequencing manipulation, generating longer read length and
with assembly mechanisms [54]. faster running time that provide better taxonomic resolution
Microarray or biochip, or gene chip are other high- [70]. Due to longer reads of PacBio sequencing, one can
throughput platforms for identifying species. The ability to sequence through longer repetitive sequences and detect
identify thousands of targets in a single hybridization experi- mutations, many of which are linked to disease. Further-
ment makes microarray one of the most potent molecular more, because of its potential to sequence full-length tran-
tools [60]. A microarray made up of a DNA barcode that scripts, it is beneficial for identifying gene isoforms and
may be used to design probe sequences in microarray analy- allows reliable discoveries of novel genes and novel iso-
sis. A DNA microarray containing a species-specific oli- forms of annotated genes. Furthermore, PacBio’s sequencing
gonucleotide probe is a viable alternative to the traditional technique can be used to detect base modification such as
Sanger sequencing for identifying species in food sample. methylation [71]. PacBio sequencing also has some draw-
Several commercial DNA chips are available to identify backs including costly, high error rate, and low throughput
animal species in food samples (e.g. CarnoCheck DNA- [71, 72]. The High sequencing error rate can be reduced
Chip, Greiner Bio-One, Austria; LCD Array Kit MEAT by re-sequencing of circular molecules several times. So
5.0, Chipron, Germany) [61]. Fish species are identified in far PacBio sequencing has been used successfully in meta-
both culinary and forensic samples using 16 S rRNA gene, barcoding analysis of arthropods and fungi [72]. Several
Cytochrome b, and COI derived probes [61–63]. Shortly, the researchers suggested that to use PacBio sequencing along
microarray-based identification approach will play a more with SGS since both of them are highly complementary in
prominent role in molecular species identification [56]. term of their advantage [70, 71].
Third generation sequencing such as Oxford Nanopore MALDI–TOF MS (Matrix-assisted laser desorption/
Technologies (ONT)’s MinION™ and PacBio sequencing ionization time of flight mass spectrometry) is being more
is an another sequencing advancement that makes DNA bar- commonly employed as a novel tool for barcoding, however
coding more feasible [64]. MinION nanopore sequencing this method should be based on accurate species identifica-
overcome the limitation associated with the Sanger sequenc- tion both morphologically and genetically. This approach
ing and NGS. Sanger sequencing is costly and requires well is extensively used to identify arthropods [73]. Other than
equipped molecular laboratory and ABI sequencer. On the arthropods, MALDI TOF MS has been successfully used in
other hand, next generation sequencing is cost-effective identification of bacteria and archaea [74].
only when large numbers of specimens are barcoded simul- DNA barcoding in combination with nanotechnology is
taneously, generate sequence reads with high accuracy, another novel approach that has been shown to be highly
also requires expensive equipment in laboratory and has sensitive, allowing for rapid uniplex and multiplex detection
long sequencing run time [65]. ONT MinION™ nanopore of pathogens in food, blood, and other samples [75]. Nano-
sequencing, introduced in 2014, is authentic, quick, third based detection methods increase the sensitivity level up to
generation sequencing, cost-effective, generate long reads, ten times as compare to PCR and other detection methods
enables real time analysis and do not require well-equipped such as radio-immunoassay, microarrays, enzyme-linked
molecular laboratory [64]. Various studies proposed that immunosorbent assay (ELISA) etc. Gold nanoparticles and
complete genome sequence of microbes can be obtained magnetic nanoparticles based “fluorescent bio-barcode DNA
by using multiplexed reads from a single MinION™ run assay” has been used to probe the Salmonella enteritidis
in combination with matched Illumina short reads such as genes [76]. Another bacterial gene Exotoxin A has been
Staphylococcus aureus, Klebsiella pneumoniae, and multid- detected by using magnetic and gold nanoparticles-based
rug resistance encoding plasmid [64, 66]. With the introduc- fluorescence bio-barcode DNA assay [77]. Recently, Ding
tion of MinION nanopore sequencing several full plasmid et al. (2021) identified the DNA marker in liquors, condi-
sequences can now be obtained in a single MinION run ments and milk by using gold nanoparticles [78]. Valen-
using a quick barcoding methodology. MinION™ has also tini et al. (2017) introduced a new approach, NanoTracer
been successfully used in bacterial and plant identification, that streamlines all the analytical steps involved with tradi-
microbiome characterisation, and DNA fingerprinting [67, tional DNA barcoding and enabling it sequencing-free and
13
accessible outside the specialized laboratories. NanoTracer data are used, even if the species is represented by a single
enables quick naked eye molecular validation of any food specimen [85].
with simple and inexpensive processing and limited instru- All of the advancements discussed above, particularly
mentation [79]. Species-specific lateral flow dipstick (LFD) HTS sequencing, whole genome sequencing, and metagen-
assays developed by Taboada et al. (2017) for identifying omics, have been viewed as a threat to DNA barcoding. HTS
Atlantic cod, Pacific cod, Alaska pollock and ling in food sequencing, whole genome sequencing, and metagenomics
products, using gold nanoparticles to enable visual identifi- produce massive amounts of genomic data. The genomic
cation with high sensitivity even for processed samples [80]. data analysis takes more time, requires more bioinformatic
expertise compared to standardized DNA barcodes, requires
more energy for data computation and storage, and is dif-
ficult to control quality when shared [86]. Therefore, DNA
Alternatives to DNA barcoding barcoding remains the preferred method for species iden-
tification and biomonitoring, while genomics is useful for
Dip-stick approach is a recent innovation in which lateral understanding genome complexity, diversity, and function.
flow assay combined with species specific primer to detect Rather than being a threat, barcoding and genomics have
wide variety of species from environmental samples [55]. clear mutual benefits, with DNA barcoding establishing a
Non-targeted NGS is an alternative to DNA barcoding platform for well-identified samples in genome sequencing
for species identification, phylogenetics, and phylogeog- projects and genomic studies contributing insights that may
raphy. Non-targeted NGS methods, such as whole genome identify new barcode regions in groups where the standard
sequencing, metagenomics and mitogenomics, do not rely regions are suboptimal [55].
on amplification. Therefore, problems like primer biases and
non-standard amplification have no effect on these methods
[81]. Reference library construction
Mitogenomics is a variant of metagenomics, shotgun
sequencing approach that uses mitochondrial genomes as Currently, DNA Barcoding (Metabarcoding) is the most
references rather than nuclear genomes. Mitogenomes are effective approach for identifying species, and its accuracy is
easily amenable to genome skimming, in which a high copy relied on the resolution of DNA barcodes and the reference
region of the genome is assembled into longer contigs from library. BOLD is the largest reference library or database and
low coverage shotgun sequencing of a specimen mixture its growth has been exponential over the last decades. The
[82]. This method is desirable because of its advantages. International Barcode of Life Consortium (iBOL) launches
Firstly, a mitogenome and its genes are commonly used several projects to expand the DNA barcode reference
molecular markers. Secondly, the mitogenomes structure library or database, including 500K (completed in 2015),
are conserved, whereas sequences can be extremely diverse. BIOSCAN (launched in 2019), and the Earth Biogenome
Thirdly, mitogenomes are small and easy to obtain and can Project [55]. Despite this, very few such libraries have been
be reconstructed directly using bioinformatics methods. developed.
Fourthly, large numbers of mitogenomes are available in Constructing a reference library with extensive species
public databases [83]. Furthermore, this approach is not coverage presents several challenges. The first challenge
affected by problems like Primer biases and non-specific is the high expense of collecting raw data, which can be
amplification. Several studies have shown that mitogenom- accomplished through DNA sequencing. Conventional
ics outperforms metabarcoding in terms of discriminatory sanger sequencing is expensive and of low efficiency [11].
power [83, 84]. However, the utility of mitogenomic is lim- This obstacle must be overcome by acquiring NGS and third
ited as it is quite expensive because each sample requires an generation sequencing platforms such as PacBio and Nanop-
individually prepared library, samples must be sequenced ore. Another challenge is selecting a critical sequencing plat-
more deeply than for metabarcoding, and assembling a form for obtaining high quality results at a low cost, which
mitogenome reference database incurs additional costs for can be accomplished by taking into consideration base qual-
specimen acquisition, sequencing, and assembly [84]. It has ity, data sizes, sequencing depth, and cost efficiency [87].
been found that phylogentics constructed on the basis of There are several NGS platforms but the most appropri-
mitogenomics or nuclear ribosomal RNA repeats are well ate choice for DNA barcoding is Roche-454 [88], which is
resolved and with this, one can distinguish between closely no longer available. In terms of high base quality and low
related species. cost, the Illumina system and Ion Torrent S5 platform are
Bayesian inference under the multispecies coalescent currently the most suitable NGS platform for conventional
model is also an alternative to DNA barcoding. This method DNA barcoding than third generation sequencing platforms
can discriminate species with high power when multi-locus [87]. Several studies have compared the performance of the
13
Illumina and Ion torrent platforms, but researchers are still This technique can also help to settle enduring nomen-
unsure which one is better suitable for DNA barcoding. Both clatural debates, leading to the taxonomic revision of
the Illumina system and the Ion Torrent S5 generate massive inadequately defined morphospecies. DNA barcoding
amounts of data, posing new challenges for data analysis approach is also widely used by ecologist due to several
[89, 90]. Several software packages have been developed, reasons. First, the diversity of ecologically essential life
including Vsearch, Usearch, Mothur, Zotu, DADA2, and forms such as ciliates and nematodes are mostly unknown
others. However, these current softwares is not perfect for and the DNA barcoding approach is a better way to assess
creating DNA barcodes, and it was not designed for con- the biodiversity of such life forms [34, 92]. Second, DNA
ventional DNA barcode data analysis. A new data analysis barcode can also detect endangered species from hair and
method called Cotu has been developed for conventional faeces sample left behind by animals [57]. Third, illegal
DNA barcodes, and its performance outperforms other com- trade in animal by-product can be monitored with the help
monly used methods like Zotu and DADA2 [87]. However, of DNA barcoding technique [93]. Fourth, DNA barcoding
more research is needed to confirm and adopt Cotu for data can be advantageous in the field of biosecurity. This is one
analysis. Using an appropriate NGS platform and advanced of the available technique to identify invasive species at a
data analysis methods, a regional or even global DNA bar- very early stage of their life cycle, such as an egg or larval
coding reference library with high species coverage is likely stage [7]. Fifth, the past environment can be reconstructed
to be developed within a few years. by using this technique. Finally, by using the DNA barcod-
The majority of current work on DNA barcoding has ing approach diet of animals can be analysed from faeces
been done in Europe and North America, which could be or stomach content [57]. Within the food industry, DNA
another reason for the limited reference library/database. barcoding reveals mislabelling of processed food that
Financial assistance is also required for the creation of a may lead to health hazards. Recently COI gene is used as
high-quality reference library. Funding for DNA barcoding a DNA barcode to reveal mislabelling of seafood in the
research should encourage the creation and curation of a European market [94]. DNA barcoding can be highly use-
reference library. A large number of national and global col- ful in forensic science [20, 57]. Some species of plants are
laborations will aid in financial support as well as to com- poisonous in nature, such as Datura sp., Brugmansia sp.,
bine local knowledge on species identification with sequenc- and Cannabis sativa, which cause serious health problems
ing capacity [91]. There are several curated natural history to humans and animals when ingested. Rapid identification
museums around the world that house a large number of of the poisonous plant is required for appropriate treat-
vouchered specimens. Obtaining DNA barcoding data from ment, and identification from vomited or excreted sam-
these vouchered specimens should significantly improve ples by visual observation is not feasible because most of
the quality of the reference database [55]. Another possible the plant part can be degraded. So, DNA barcoding will
step would be to incorporate reference barcodes on a regu- be useful for identification from these degraded samples.
lar basis. To improve the reference barcode library, make it Recently rbcL and ITS2 genes are used as a barcoding
mandatory to submit the reference barcode when describing marker for identifying poisonous plant species [20].
a new species. DNA barcoding tool overcomes the limitation of the
classical identification method, but this approach itself has
certain restrictions. One of the most significant drawbacks
Strength and limitation of DNA barcoding of the DNA barcoding method is that there is no universal
primer or universal gene found in all forms of life and has
Apart from taxonomists, the DNA barcoding technique enough sequence divergence to allow for species differen-
can benefit scientists from other fields such as biotech- tiation [56]. Very less number of reference DNA barcode
nology, food industries, forensic science, and animal diet library, and Loss of quantitative information due to primer
[57]. Taxonomist uses a sensu-stricto (refers to the identi- and polymerase biases [84]. DNA barcoding distinguishes
fication of species level using a single standardized DNA species based on intraspecific and interspecific genetic vari-
fragment) approach of DNA barcoding, while other sci- ation, although the ranges of such variation are unclear and
entists use a sensu-lato (refers to the identification of any may differ between taxa [31]. The existence of pseudogenes
taxonomic group using any DNA fragment) approach. The and heteroplasmy reduces the accuracy of DNA barcoding
main application of DNA barcode in taxonomy to acceler- and increases the complexity of database. A pseudogene can
ate the species identification and revealing cryptic species. result in the erroneous division of single species into sev-
DNA barcode data can provide a comprehensive founda- eral species. Pseudogenes can produce heteroplasmy, which
tion for organizing and identifying species-rich groups in causes more than one kind of mtDNA to coexist in the same
the tree of life, serving as a good starting point for tax- individual and limiting species identification by DNA bar-
onomy, biodiversity assessments, and biomonitoring [55]. coding [56].
13
Conclusion Consent to publish Not applicable.
Through the rapid development in the last 2 decades, DNA

barcoding has emerged as a highly effective molecular tool
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Molecular Biology Reports (2023) 50:761–775