scholarly journals MinION-Based DNA Barcoding of Preserved and Non-Invasively Collected Wildlife Samples

Genes ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 445 ◽  
Author(s):  
Adeline Seah ◽  
Marisa C.W. Lim ◽  
Denise McAloose ◽  
Stefan Prost ◽  
Tracie A. Seimon
Keyword(s):  
Dna Barcoding ◽  
Species Identification ◽  
Wildlife Conservation ◽  
Sequence Similarity ◽  
Dna Barcode ◽  
Bioinformatics Pipeline ◽  
Tissue Samples ◽  
Consensus Sequences ◽  
Sequencing Errors ◽  
Fresh Frozen

The ability to sequence a variety of wildlife samples with portable, field-friendly equipment will have significant impacts on wildlife conservation and health applications. However, the only currently available field-friendly DNA sequencer, the MinION by Oxford Nanopore Technologies, has a high error rate compared to standard laboratory-based sequencing platforms and has not been systematically validated for DNA barcoding accuracy for preserved and non-invasively collected tissue samples. We tested whether various wildlife sample types, field-friendly methods, and our clustering-based bioinformatics pipeline, SAIGA, can be used to generate consistent and accurate consensus sequences for species identification. Here, we systematically evaluate variation in cytochrome b sequences amplified from scat, hair, feather, fresh frozen liver, and formalin-fixed paraffin-embedded (FFPE) liver. Each sample was processed by three DNA extraction protocols. For all sample types tested, the MinION consensus sequences matched the Sanger references with 99.29%–100% sequence similarity, even for samples that were difficult to amplify, such as scat and FFPE tissue extracted with Chelex resin. Sequencing errors occurred primarily in homopolymer regions, as identified in previous MinION studies. We demonstrate that it is possible to generate accurate DNA barcode sequences from preserved and non-invasively collected wildlife samples using portable MinION sequencing, creating more opportunities to apply portable sequencing technology for species identification.

scholarly journals MinION-based DNA barcoding of preserved and non-invasively collected wildlife samples

2020 ◽  
Author(s):  
Adeline Seah ◽  
Marisa C.W. Lim ◽  
Denise McAloose ◽  
Stefan Prost ◽  
Tracie Seimon
Keyword(s):  
Dna Barcoding ◽  
Species Identification ◽  
Wildlife Conservation ◽  
Sequence Similarity ◽  
Dna Barcode ◽  
List Type ◽  
Bioinformatics Pipeline ◽  
Tissue Samples ◽  
Consensus Sequences ◽  
Sequencing Errors

AbstractThe ability to sequence a variety of wildlife samples with portable, field-friendly equipment will have significant impacts on wildlife conservation and health applications. However, the only currently available field-friendly DNA sequencer, the MinION by Oxford Nanopore Technologies, has a high error rate compared to standard laboratory-based sequencing platforms and has not been systematically validated for DNA barcoding accuracy for preserved and non-invasively collected tissue samples.We tested whether various wildlife sample types, field-friendly methods, and our clustering-based bioinformatics pipeline, SAIGA, can be used to generate consistent and accurate consensus sequences for species identification. Here, we systematically evaluate variation in cytochrome b sequences amplified from scat, hair, feather, fresh frozen liver, and formalin-fixed paraffin-embedded (FFPE) liver. Each sample was processed by three DNA extraction protocols.For all sample types tested, the MinION consensus sequences matched the Sanger references with 99.29-100% sequence similarity, even for samples that were difficult to amplify, such as scat and FFPE tissue extracted with Chelex resin. Sequencing errors occurred primarily in homopolymer regions, as identified in previous MinION studies.We demonstrate that it is possible to generate accurate DNA barcode sequences from preserved and non-invasively collected wildlife samples using portable MinION sequencing, creating more opportunities to apply portable sequencing technology for species identification.

scholarly journals Increased yields of duplex sequencing data by a series of quality control tools

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Gundula Povysil ◽  
Monika Heinzl ◽  
Renato Salazar ◽  
Nicholas Stoler ◽  
Anton Nekrutenko ◽  
...  
Keyword(s):  
Low Frequency ◽  
Variant Calling ◽  
Data Loss ◽  
Sequencing Data ◽  
Bioinformatics Pipeline ◽  
Consensus Sequences ◽  
Sequencing Errors ◽  
Data Output ◽  
Reverse Strand ◽  
Duplex Sequencing

Abstract Duplex sequencing is currently the most reliable method to identify ultra-low frequency DNA variants by grouping sequence reads derived from the same DNA molecule into families with information on the forward and reverse strand. However, only a small proportion of reads are assembled into duplex consensus sequences (DCS), and reads with potentially valuable information are discarded at different steps of the bioinformatics pipeline, especially reads without a family. We developed a bioinformatics toolset that analyses the tag and family composition with the purpose to understand data loss and implement modifications to maximize the data output for the variant calling. Specifically, our tools show that tags contain polymerase chain reaction and sequencing errors that contribute to data loss and lower DCS yields. Our tools also identified chimeras, which likely reflect barcode collisions. Finally, we also developed a tool that re-examines variant calls from raw reads and provides different summary data that categorizes the confidence level of a variant call by a tier-based system. With this tool, we can include reads without a family and check the reliability of the call, that increases substantially the sequencing depth for variant calling, a particular important advantage for low-input samples or low-coverage regions.

Research Paper DNA barcode identification of fish products from Guiyang markets in southwest China

2022 ◽  
Author(s):  
Qian Tang ◽  
Qi Luo ◽  
Qian Duan ◽  
Lei Deng ◽  
Renyi Zhang
Keyword(s):  
Dna Barcoding ◽  
Molecular Identification ◽  
Fish Consumption ◽  
Southwest China ◽  
Dna Barcode ◽  
Guizhou Province ◽  
Accurate Identification ◽  
Continuous Growth ◽  
Fish Products ◽  
Fresh Frozen

Nowadays, the global fish consumption continues to rise along with the continuous growth of the population, which has led to the dilemma of overfishing of fishery resources. Especially high-value fish that are overfished are often replaced by other fish. Therefore, the accurate identification of fish products in the market is a problem worthy of attention. In this study, full-DNA barcoding (FDB) and mini-DNA barcoding (MDB) used to detect the fraud of fish products in Guiyang, Guizhou province in China. The molecular identification results showed that 39 of the 191 samples were not consistent with the labels. The mislabelling of fish products for fresh, frozen, cooked and canned were 11.70%, 20.00%, 34.09% and 50.00%, respectively. The average kimura 2 parameter distances of MDB within species and genera were 0.27% and 5.41%, respectively; while average distances of FDB were 0.17% within species and 6.17% within genera. In this study, commercial fraud is noticeable, most of the high-priced fish were replaced of low-priced fish with a similar feature. Our study indicated that DNA barcoding is a valid tool for the identification of fish products and that it allows an idea of conservation and monitoring efforts, while confirming the MDB as a reliable tool for fish products.

scholarly journals Assessing universality of DNA barcoding in geographically isolated selected desert medicinal species of Fabaceae and Poaceae

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4499 ◽  
Author(s):  
Aisha Tahir ◽  
Fatma Hussain ◽  
Nisar Ahmed ◽  
Abdolbaset Ghorbani ◽  
Amer Jamil
Keyword(s):  
Dna Barcoding ◽  
Species Identification ◽  
Phylogenetic Trees ◽  
Sequence Similarity ◽  
Sequence Divergence ◽  
Species Level ◽  
Practical Implementation ◽  
Medicinal Species ◽  
Level Identification ◽  
Geographically Isolated

In pursuit of developing fast and accurate species-level molecular identification methods, we tested six DNA barcodes, namely ITS2, matK, rbcLa, ITS2+matK, ITS2+rbcLa, matK+rbcLa and ITS2+matK+rbcLa, for their capacity to identify frequently consumed but geographically isolated medicinal species of Fabaceae and Poaceae indigenous to the desert of Cholistan. Data were analysed by BLASTn sequence similarity, pairwise sequence divergence in TAXONDNA, and phylogenetic (neighbour-joining and maximum-likelihood trees) methods. Comparison of six barcode regions showed that ITS2 has the highest number of variable sites (209/360) for tested Fabaceae and (106/365) Poaceae species, the highest species-level identification (40%) in BLASTn procedure, distinct DNA barcoding gap, 100% correct species identification in BM and BCM functions of TAXONDNA, and clear cladding pattern with high nodal support in phylogenetic trees in both families. ITS2+matK+rbcLa followed ITS2 in its species-level identification capacity. The study was concluded with advocating the DNA barcoding as an effective tool for species identification and ITS2 as the best barcode region in identifying medicinal species of Fabaceae and Poaceae. Current research has practical implementation potential in the fields of pharmaco-vigilance, trade of medicinal plants and biodiversity conservation.

scholarly journals Multi-locus DNA barcoding identifies matK as a suitable marker for species identification in Hibiscus L.

Genome ◽  
2016 ◽  
Vol 59 (12) ◽  
pp. 1150-1156 ◽  
Author(s):  
Sundar Poovitha ◽  
Nithaniyal Stalin ◽  
Raju Balaji ◽  
Madasamy Parani
Keyword(s):  
Dna Barcoding ◽  
Species Identification ◽  
Dna Barcode ◽  
Species Differentiation ◽  
Molecular Method ◽  
Peninsular India ◽  
Plant Materials ◽  
Medicinal Value ◽  
Formed Part ◽  
Suitable Marker

The genus Hibiscus L. includes several taxa of medicinal value and species used for the extraction of natural dyes. These applications require the use of authentic plant materials. DNA barcoding is a molecular method for species identification, which helps in reliable authentication by using one or more DNA barcode marker. In this study, we have collected 44 accessions, representing 16 species of Hibiscus, distributed in the southern peninsular India, to evaluate the discriminatory power of the two core barcodes rbcLa and matK together with the suggested additional regions trnH-psbA and ITS2. No intraspecies divergence was observed among the accessions studied. Interspecies divergence was 0%–9.6% with individual markers, which increased to 0%–12.5% and 0.8%–20.3% when using two- and three-marker combinations, respectively. Differentiation of all the species of Hibiscus was possible with the matK DNA barcode marker. Also, in two-marker combinations, only those combinations with matK differentiated all the species. Though all the three-marker combinations showed 100% species differentiation, species resolution was consistently better when the matK marker formed part of the combination. These results clearly showed that matK is more suitable when compared to rbcLa, trnH-psbA, and ITS2 for species identification in Hibiscus.

scholarly journals Identification of species adulteration in traded medicinal plant raw drugs using DNA barcoding

Genome ◽  
2017 ◽  
Vol 60 (2) ◽  
pp. 139-146 ◽  
Author(s):  
Stalin Nithaniyal ◽  
Sophie Lorraine Vassou ◽  
Sundar Poovitha ◽  
Balaji Raju ◽  
Madasamy Parani
Keyword(s):  
Medicinal Plants ◽  
Medicinal Plant ◽  
Alternative Medicine ◽  
Dna Barcoding ◽  
Species Identification ◽  
Dna Barcode ◽  
Regulatory Agencies ◽  
Vernacular Names ◽  
Identification Of Species ◽  
Whole Plants

Plants are the major source of therapeutic ingredients in complementary and alternative medicine (CAM). However, species adulteration in traded medicinal plant raw drugs threatens the reliability and safety of CAM. Since morphological features of medicinal plants are often not intact in the raw drugs, DNA barcoding was employed for species identification. Adulteration in 112 traded raw drugs was tested after creating a reference DNA barcode library consisting of 1452 rbcL and matK barcodes from 521 medicinal plant species. Species resolution of this library was 74.4%, 90.2%, and 93.0% for rbcL, matK, and rbcL + matK, respectively. DNA barcoding revealed adulteration in about 20% of the raw drugs, and at least 6% of them were derived from plants with completely different medicinal or toxic properties. Raw drugs in the form of dried roots, powders, and whole plants were found to be more prone to adulteration than rhizomes, fruits, and seeds. Morphological resemblance, co-occurrence, mislabeling, confusing vernacular names, and unauthorized or fraudulent substitutions might have contributed to species adulteration in the raw drugs. Therefore, this library can be routinely used to authenticate traded raw drugs for the benefit of all stakeholders: traders, consumers, and regulatory agencies.

scholarly journals DNA Barcoding Analysis and Phylogenetic Relation of Mangroves in Guangdong Province, China

Forests ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 56 ◽  
Author(s):  
Feng Wu ◽  
Mei Li ◽  
Baowen Liao ◽  
Xin Shi ◽  
Yong Xu
Keyword(s):  
Dna Barcoding ◽  
Species Identification ◽  
Dna Barcode ◽  
Phylogenetic Reconstruction ◽  
Pcr Amplification ◽  
Guangdong Province ◽  
Nuclear Ribosomal Dna ◽  
Success Rates ◽  
Mangrove Species ◽  
Identification Rate

Mangroves are distributed in the transition zone between sea and land, mostly in tropical and subtropical areas. They provide important ecosystem services and are therefore economically valuable. DNA barcoding is a useful tool for species identification and phylogenetic reconstruction. To evaluate the effectiveness of DNA barcoding in identifying mangrove species, we sampled 135 individuals representing 23 species, 22 genera, and 17 families from Zhanjiang, Shenzhen, Huizhou, and Shantou in the Guangdong province, China. We tested the universality of four DNA barcodes, namely rbcL, matK, trnH-psbA, and the internal transcribed spacer of nuclear ribosomal DNA (ITS), and examined their efficacy for species identification and the phylogenetic reconstruction of mangroves. The success rates for PCR amplification of rbcL, matK, trnH-psbA, and ITS were 100%, 80.29% ± 8.48%, 99.38% ± 1.25%, and 97.18% ± 3.25%, respectively, and the rates of DNA sequencing were 100%, 75.04% ± 6.26%, 94.57% ± 5.06%, and 83.35% ± 4.05%, respectively. These results suggest that both rbcL and trnH–psbA are universal in mangrove species from the Guangdong province. The highest success rate for species identification was 84.48% ± 12.09% with trnH-psbA, followed by rbcL (82.16% ± 9.68%), ITS (66.48% ± 5.97%), and matK (65.09% ± 6.00%), which increased to 91.25% ± 9.78% with the addition of rbcL. Additionally, the identification rate of mangroves was not significantly different between rbcL + trnH-psbA and other random fragment combinations. In conclusion, rbcL and trnH-psbA were the most suitable DNA barcode fragments for species identification in mangrove plants. When the phylogenetic relationships were constructed with random fragment combinations, the optimal evolutionary tree with high supporting values (86.33% ± 4.16%) was established using the combination of matK + rbcL + trnH-psbA + ITS in mangroves. In total, the 476 newly acquired sequences in this study lay the foundation for a DNA barcode database of mangroves.

scholarly journals DNA barcoding British Euphrasia reveals deeply divergent polyploids but lack of species-level resolution

2017 ◽  
Author(s):  
Xumei Wang ◽  
Galina Gussarova ◽  
Markus Ruhsam ◽  
Natasha de Vere ◽  
Chris Metherell ◽  
...  
Keyword(s):  
Genetic Variation ◽  
Dna Barcoding ◽  
Species Identification ◽  
Dna Barcode ◽  
Species Level ◽  
Ecological Processes ◽  
Evolutionary Patterns ◽  
Ploidy Levels ◽  
Self Fertilization ◽  
Phylogenetic Framework

ABSTRACTBackground and aimsDNA barcoding is emerging as a useful tool not only for species identification but for studying evolutionary and ecological processes. Although plant DNA barcodes do not always provide species-level resolution, the generation of large DNA barcode datasets can provide insights into the mechanisms underlying the generation of species diversity. Here, we use DNA barcoding to study evolutionary processes in taxonomically complex British Euphrasia, a group with multiple ploidy levels, frequent self- fertilization, young species divergence and widespread hybridisation.MethodsWe sequenced the core plant barcoding loci, supplemented with additional nuclear and plastid loci, in representatives of all 19 British Euphrasia species. We analyse these data in a population genetic and phylogenetic framework. We then date the divergence of haplotypes in a global Euphrasia dataset using a time-calibrated Bayesian approach implemented in BEAST.Key resultsNo Euphrasia species has a consistent diagnostic haplotype. Instead, haplotypes are either widespread across species, or are population specific. Nuclear genetic variation is strongly partitioned by ploidy levels, with diploid and tetraploid British Euphrasia possessing deeply divergent ITS haplotypes (DXY = 5.1%), with haplotype divergence corresponding to the late Miocene. In contrast, plastid data show no clear division by ploidy, and instead reveal weakly supported geographic patterns.ConclusionsUsing standard DNA barcoding loci for species identification in Euphrasia will be unsuccessful. However, these loci provide key insights into the maintenance of genetic variation, with divergence of diploids and tetraploids suggesting that ploidy differences act as a barrier to gene exchange in British Euphrasia, with rampant hybridisation within ploidy levels. The scarcity of shared diploid-tetraploid ITS haplotypes supports the polyploids being allotetraploid in origin. Overall, these results show that even when lacking species-level resolution, DNA barcoding can reveal insightful evolutionary patterns in taxonomically complex genera.

Testing the Effectiveness of DNA Barcoding for Biodiversity Assessment of Moths from Nigeria

Diversity ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 85
Author(s):  
Lotanna Micah Nneji ◽  
Adeniyi Charles Adeola ◽  
Yun-Yu Wang ◽  
Adeyemi Mufutau Ajao ◽  
Okorie Anyaele ◽  
...  
Keyword(s):  
Maximum Likelihood ◽  
Dna Barcoding ◽  
Species Identification ◽  
Phylogenetic Analyses ◽  
Dna Barcode ◽  
Molecular Data ◽  
Population Divergence ◽  
Biodiversity Assessment ◽  
Taxonomic Methods ◽  
Future Work

Comprehensive biodiversity assessment of moths in Nigeria rely greatly on accurate species identification. While most of the Nigerian moths are identified effortlessly using their morphological traits, some taxa are morphologically indistinguishable, which makes it difficult for taxon diagnosis. We investigated the efficiency of the DNA barcode, a fragment of the mitochondrial Cytochrome C oxidase subunit I, as a tool for the identification of Nigerian moths. We barcoded 152 individuals comprising 18 morphospecies collected from one of the remaining and threatened rainforest blocks of Nigeria – the Cross River National Park. Phenetic neighbor-joining tree and phylogenetic Maximum Likelihood approach were employed for the molecular-based species identification. Results showed that DNA barcodes enabled species-level identification of most of the individuals collected from the Park. Additionally, DNA barcoding unraveled the presence of at least six potential new and yet undescribed species—Amnemopsyche sp., Arctia sp., Deinypena sp., Hodebertia sp., Otroeda sp., and Palpita sp. The phylogenetic Maximum Likelihood using the combined dataset of all the newly assembled sequences from Nigeria showed that all species formed unique clades. The phylogenetic analyses provided evidence of population divergence in Euchromia lethe, Nyctemera leuconoe, and Deinypena lacista. This study thus illustrates the efficacy of DNA barcoding for species identification and discovery of potential new species, which demonstrates its relevance in biodiversity documentation of Nigerian moths. Future work should, therefore, extend to the creation of an exhaustive DNA barcode reference library comprising all species of moths from Nigeria to have a comprehensive insight on the diversity of moths in the country. Finally, we propose integrated taxonomic methods that would combine morphological, ecological, and molecular data in the identification and diversity studies of moths in Nigeria.

DNA barcoding as an aid for species identification in austral black flies (Insecta: Diptera: Simuliidae)

Genome ◽  
2017 ◽  
Vol 60 (4) ◽  
pp. 348-357 ◽  
Author(s):  
Luis M. Hernández-Triana ◽  
Fernanda Montes De Oca ◽  
Sean W.J. Prosser ◽  
Paul D.N. Hebert ◽  
T. Ryan Gregory ◽  
...  
Keyword(s):  
Dna Barcoding ◽  
Species Identification ◽  
Dna Barcode ◽  
Distinct Species ◽  
Coi Gene ◽  
Black Flies ◽  
Cryptic Diversity ◽  
Dna Barcodes ◽  
Identification Of Species ◽  
Species Groups

In this paper, the utility of a partial sequence of the COI gene, the DNA barcoding region, for the identification of species of black flies in the austral region was assessed. Twenty-eight morphospecies were analyzed: eight of the genus Austrosimulium (four species in the subgenus Austrosimulium s. str., three species in the subgenus Novaustrosimulium, and one species unassigned to subgenus), two of the genus Cnesia, eight of Gigantodax, three of Paracnephia, one of Paraustrosimulium, and six of Simulium (subgenera Morops, Nevermannia, and Pternaspatha). The neighbour-joining tree derived from the DNA barcode sequences grouped most specimens according to species or species groups recognized by morphotaxonomic studies. Intraspecific sequence divergences within morphologically distinct species ranged from 0% to 1.8%, while higher divergences (2%–4.2%) in certain species suggested the presence of cryptic diversity. The existence of well-defined groups within S. simile revealed the likely inclusion of cryptic diversity. DNA barcodes also showed that specimens identified as C. dissimilis, C. nr. pussilla, and C. ornata might be conspecific, suggesting possible synonymy. DNA barcoding combined with a sound morphotaxonomic framework would provide an effective approach for the identification of black flies in the region.

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