DNA barcode authentication of wood samples of threatened and commercial timber trees within the tropical dry evergreen forest of India.

PubMed

Nithaniyal, Stalin; Newmaster, Steven G; Ragupathy, Subramanyam; Krishnamoorthy, Devanathan; Vassou, Sophie Lorraine; Parani, Madasamy

2014-01-01

India is rich with biodiversity, which includes a large number of endemic, rare and threatened plant species. Previous studies have used DNA barcoding to inventory species for applications in biodiversity monitoring, conservation impact assessment, monitoring of illegal trading, authentication of traded medicinal plants etc. This is the first tropical dry evergreen forest (TDEF) barcode study in the World and the first attempt to assemble a reference barcode library for the trees of India as part of a larger project initiated by this research group. We sampled 429 trees representing 143 tropical dry evergreen forest (TDEF) species, which included 16 threatened species. DNA barcoding was completed using rbcL and matK markers. The tiered approach (1st tier rbcL; 2nd tier matK) correctly identified 136 out of 143 species (95%). This high level of species resolution was largely due to the fact that the tree species were taxonomically diverse in the TDEF. Ability to resolve taxonomically diverse tree species of TDEF was comparable among the best match method, the phylogenetic method, and the characteristic attribute organization system method. We demonstrated the utility of the TDEF reference barcode library to authenticate wood samples from timber operations in the TDEF. This pilot research study will enable more comprehensive surveys of the illegal timber trade of threatened species in the TDEF. This TDEF reference barcode library also contains trees that have medicinal properties, which could be used to monitor unsustainable and indiscriminate collection of plants from the wild for their medicinal value.

DNA Barcode Authentication of Wood Samples of Threatened and Commercial Timber Trees within the Tropical Dry Evergreen Forest of India

PubMed Central

Nithaniyal, Stalin; Newmaster, Steven G.; Ragupathy, Subramanyam; Krishnamoorthy, Devanathan; Vassou, Sophie Lorraine; Parani, Madasamy

2014-01-01

Background India is rich with biodiversity, which includes a large number of endemic, rare and threatened plant species. Previous studies have used DNA barcoding to inventory species for applications in biodiversity monitoring, conservation impact assessment, monitoring of illegal trading, authentication of traded medicinal plants etc. This is the first tropical dry evergreen forest (TDEF) barcode study in the World and the first attempt to assemble a reference barcode library for the trees of India as part of a larger project initiated by this research group. Methodology/Principal Findings We sampled 429 trees representing 143 tropical dry evergreen forest (TDEF) species, which included 16 threatened species. DNA barcoding was completed using rbcL and matK markers. The tiered approach (1st tier rbcL; 2nd tier matK) correctly identified 136 out of 143 species (95%). This high level of species resolution was largely due to the fact that the tree species were taxonomically diverse in the TDEF. Ability to resolve taxonomically diverse tree species of TDEF was comparable among the best match method, the phylogenetic method, and the characteristic attribute organization system method. Conclusions We demonstrated the utility of the TDEF reference barcode library to authenticate wood samples from timber operations in the TDEF. This pilot research study will enable more comprehensive surveys of the illegal timber trade of threatened species in the TDEF. This TDEF reference barcode library also contains trees that have medicinal properties, which could be used to monitor unsustainable and indiscriminate collection of plants from the wild for their medicinal value. PMID:25259794

Identifying insects with incomplete DNA barcode libraries, African fruit flies (Diptera: Tephritidae) as a test case.

PubMed

Virgilio, Massimiliano; Jordaens, Kurt; Breman, Floris C; Backeljau, Thierry; De Meyer, Marc

2012-01-01

We propose a general working strategy to deal with incomplete reference libraries in the DNA barcoding identification of species. Considering that (1) queries with a large genetic distance with their best DNA barcode match are more likely to be misidentified and (2) imposing a distance threshold profitably reduces identification errors, we modelled relationships between identification performances and distance thresholds in four DNA barcode libraries of Diptera (n = 4270), Lepidoptera (n = 7577), Hymenoptera (n = 2067) and Tephritidae (n = 602 DNA barcodes). In all cases, more restrictive distance thresholds produced a gradual increase in the proportion of true negatives, a gradual decrease of false positives and more abrupt variations in the proportions of true positives and false negatives. More restrictive distance thresholds improved precision, yet negatively affected accuracy due to the higher proportions of queries discarded (viz. having a distance query-best match above the threshold). Using a simple linear regression we calculated an ad hoc distance threshold for the tephritid library producing an estimated relative identification error <0.05. According to the expectations, when we used this threshold for the identification of 188 independently collected tephritids, less than 5% of queries with a distance query-best match below the threshold were misidentified. Ad hoc thresholds can be calculated for each particular reference library of DNA barcodes and should be used as cut-off mark defining whether we can proceed identifying the query with a known estimated error probability (e.g. 5%) or whether we should discard the query and consider alternative/complementary identification methods.

Identifying Insects with Incomplete DNA Barcode Libraries, African Fruit Flies (Diptera: Tephritidae) as a Test Case

PubMed Central

Virgilio, Massimiliano; Jordaens, Kurt; Breman, Floris C.; Backeljau, Thierry; De Meyer, Marc

2012-01-01

We propose a general working strategy to deal with incomplete reference libraries in the DNA barcoding identification of species. Considering that (1) queries with a large genetic distance with their best DNA barcode match are more likely to be misidentified and (2) imposing a distance threshold profitably reduces identification errors, we modelled relationships between identification performances and distance thresholds in four DNA barcode libraries of Diptera (n = 4270), Lepidoptera (n = 7577), Hymenoptera (n = 2067) and Tephritidae (n = 602 DNA barcodes). In all cases, more restrictive distance thresholds produced a gradual increase in the proportion of true negatives, a gradual decrease of false positives and more abrupt variations in the proportions of true positives and false negatives. More restrictive distance thresholds improved precision, yet negatively affected accuracy due to the higher proportions of queries discarded (viz. having a distance query-best match above the threshold). Using a simple linear regression we calculated an ad hoc distance threshold for the tephritid library producing an estimated relative identification error <0.05. According to the expectations, when we used this threshold for the identification of 188 independently collected tephritids, less than 5% of queries with a distance query-best match below the threshold were misidentified. Ad hoc thresholds can be calculated for each particular reference library of DNA barcodes and should be used as cut-off mark defining whether we can proceed identifying the query with a known estimated error probability (e.g. 5%) or whether we should discard the query and consider alternative/complementary identification methods. PMID:22359600

R-Syst::diatom: an open-access and curated barcode database for diatoms and freshwater monitoring.

PubMed

Rimet, Frédéric; Chaumeil, Philippe; Keck, François; Kermarrec, Lenaïg; Vasselon, Valentin; Kahlert, Maria; Franc, Alain; Bouchez, Agnès

2016-01-01

Diatoms are micro-algal indicators of freshwater pollution. Current standardized methodologies are based on microscopic determinations, which is time consuming and prone to identification uncertainties. The use of DNA-barcoding has been proposed as a way to avoid these flaws. Combining barcoding with next-generation sequencing enables collection of a large quantity of barcodes from natural samples. These barcodes are identified as certain diatom taxa by comparing the sequences to a reference barcoding library using algorithms. Proof of concept was recently demonstrated for synthetic and natural communities and underlined the importance of the quality of this reference library. We present an open-access and curated reference barcoding database for diatoms, called R-Syst::diatom, developed in the framework of R-Syst, the network of systematic supported by INRA (French National Institute for Agricultural Research), see http://www.rsyst.inra.fr/en. R-Syst::diatom links DNA-barcodes to their taxonomical identifications, and is dedicated to identify barcodes from natural samples. The data come from two sources, a culture collection of freshwater algae maintained in INRA in which new strains are regularly deposited and barcoded and from the NCBI (National Center for Biotechnology Information) nucleotide database. Two kinds of barcodes were chosen to support the database: 18S (18S ribosomal RNA) and rbcL (Ribulose-1,5-bisphosphate carboxylase/oxygenase), because of their efficiency. Data are curated using innovative (Declic) and classical bioinformatic tools (Blast, classical phylogenies) and up-to-date taxonomy (Catalogues and peer reviewed papers). Every 6 months R-Syst::diatom is updated. The database is available through the R-Syst microalgae website (http://www.rsyst.inra.fr/) and a platform dedicated to next-generation sequencing data analysis, virtual_BiodiversityL@b (https://galaxy-pgtp.pierroton.inra.fr/). We present here the content of the library regarding the number of barcodes and diatom taxa. In addition to these information, morphological features (e.g. biovolumes, chloroplasts…), life-forms (mobility, colony-type) or ecological features (taxa preferenda to pollution) are indicated in R-Syst::diatom. Database URL: http://www.rsyst.inra.fr/. © The Author(s) 2016. Published by Oxford University Press.

R-Syst::diatom: an open-access and curated barcode database for diatoms and freshwater monitoring

PubMed Central

Rimet, Frédéric; Chaumeil, Philippe; Keck, François; Kermarrec, Lenaïg; Vasselon, Valentin; Kahlert, Maria; Franc, Alain; Bouchez, Agnès

2016-01-01

Diatoms are micro-algal indicators of freshwater pollution. Current standardized methodologies are based on microscopic determinations, which is time consuming and prone to identification uncertainties. The use of DNA-barcoding has been proposed as a way to avoid these flaws. Combining barcoding with next-generation sequencing enables collection of a large quantity of barcodes from natural samples. These barcodes are identified as certain diatom taxa by comparing the sequences to a reference barcoding library using algorithms. Proof of concept was recently demonstrated for synthetic and natural communities and underlined the importance of the quality of this reference library. We present an open-access and curated reference barcoding database for diatoms, called R-Syst::diatom, developed in the framework of R-Syst, the network of systematic supported by INRA (French National Institute for Agricultural Research), see http://www.rsyst.inra.fr/en. R-Syst::diatom links DNA-barcodes to their taxonomical identifications, and is dedicated to identify barcodes from natural samples. The data come from two sources, a culture collection of freshwater algae maintained in INRA in which new strains are regularly deposited and barcoded and from the NCBI (National Center for Biotechnology Information) nucleotide database. Two kinds of barcodes were chosen to support the database: 18S (18S ribosomal RNA) and rbcL (Ribulose-1,5-bisphosphate carboxylase/oxygenase), because of their efficiency. Data are curated using innovative (Declic) and classical bioinformatic tools (Blast, classical phylogenies) and up-to-date taxonomy (Catalogues and peer reviewed papers). Every 6 months R-Syst::diatom is updated. The database is available through the R-Syst microalgae website (http://www.rsyst.inra.fr/) and a platform dedicated to next-generation sequencing data analysis, virtual_BiodiversityL@b (https://galaxy-pgtp.pierroton.inra.fr/). We present here the content of the library regarding the number of barcodes and diatom taxa. In addition to these information, morphological features (e.g. biovolumes, chloroplasts…), life-forms (mobility, colony-type) or ecological features (taxa preferenda to pollution) are indicated in R-Syst::diatom. Database URL: http://www.rsyst.inra.fr/ PMID:26989149

Critical factors for assembling a high volume of DNA barcodes

PubMed Central

Hajibabaei, Mehrdad; deWaard, Jeremy R; Ivanova, Natalia V; Ratnasingham, Sujeevan; Dooh, Robert T; Kirk, Stephanie L; Mackie, Paula M; Hebert, Paul D.N

2005-01-01

Large-scale DNA barcoding projects are now moving toward activation while the creation of a comprehensive barcode library for eukaryotes will ultimately require the acquisition of some 100 million barcodes. To satisfy this need, analytical facilities must adopt protocols that can support the rapid, cost-effective assembly of barcodes. In this paper we discuss the prospects for establishing high volume DNA barcoding facilities by evaluating key steps in the analytical chain from specimens to barcodes. Alliances with members of the taxonomic community represent the most effective strategy for provisioning the analytical chain with specimens. The optimal protocols for DNA extraction and subsequent PCR amplification of the barcode region depend strongly on their condition, but production targets of 100K barcode records per year are now feasible for facilities working with compliant specimens. The analysis of museum collections is currently challenging, but PCR cocktails that combine polymerases with repair enzyme(s) promise future success. Barcode analysis is already a cost-effective option for species identification in some situations and this will increasingly be the case as reference libraries are assembled and analytical protocols are simplified. PMID:16214753

Plans and progress for building a Great Lakes fauna DNA barcode reference library

EPA Science Inventory

DNA reference libraries provide researchers with an important tool for assessing regional biodiversity by allowing unknown genetic sequences to be assigned identities, while also providing a means for taxonomists to validate identifications. Expanding the representation of Great...

DNA Barcoding for Species Identification of Insect Skins: A Test on Chironomidae (Diptera) Pupal Exuviae

PubMed Central

Ekrem, Torbjørn; Stur, Elisabeth

2017-01-01

Abstract Chironomidae (Diptera) pupal exuviae samples are commonly used for biological monitoring of aquatic habitats. DNA barcoding has proved useful for species identification of chironomid life stages containing cellular tissue, but the barcoding success of chironomid pupal exuviae is unknown. We assessed whether standard DNA barcoding could be efficiently used for species identification of chironomid pupal exuviae when compared with morphological techniques and if there were differences in performance between temperate and tropical ecosystems, subfamilies, and tribes. PCR, sequence, and identification success differed significantly between geographic regions and taxonomic groups. For Norway, 27 out of 190 (14.2%) of pupal exuviae resulted in high-quality chironomid sequences that match species. For Costa Rica, 69 out of 190 (36.3%) Costa Rican pupal exuviae resulted in high-quality sequences, but none matched known species. Standard DNA barcoding of chironomid pupal exuviae had limited success in species identification of unknown specimens due to contaminations and lack of matching references in available barcode libraries, especially from Costa Rica. Therefore, we recommend future biodiversity studies that focus their efforts on understudied regions, to simultaneously use morphological and molecular identification techniques to identify all life stages of chironomids and populate the barcode reference library with identified sequences.

When species matches are unavailable are DNA barcodes correctly assigned to higher taxa? An assessment using sphingid moths

PubMed Central

2011-01-01

Background When a specimen belongs to a species not yet represented in DNA barcode reference libraries there is disagreement over the effectiveness of using sequence comparisons to assign the query accurately to a higher taxon. Library completeness and the assignment criteria used have been proposed as critical factors affecting the accuracy of such assignments but have not been thoroughly investigated. We explored the accuracy of assignments to genus, tribe and subfamily in the Sphingidae, using the almost complete global DNA barcode reference library (1095 species) available for this family. Costa Rican sphingids (118 species), a well-documented, diverse subset of the family, with each of the tribes and subfamilies represented were used as queries. We simulated libraries with different levels of completeness (10-100% of the available species), and recorded assignments (positive or ambiguous) and their accuracy (true or false) under six criteria. Results A liberal tree-based criterion assigned 83% of queries accurately to genus, 74% to tribe and 90% to subfamily, compared to a strict tree-based criterion, which assigned 75% of queries accurately to genus, 66% to tribe and 84% to subfamily, with a library containing 100% of available species (but excluding the species of the query). The greater number of true positives delivered by more relaxed criteria was negatively balanced by the occurrence of more false positives. This effect was most sharply observed with libraries of the lowest completeness where, for example at the genus level, 32% of assignments were false positives with the liberal criterion versus < 1% when using the strict. We observed little difference (< 8% using the liberal criterion) however, in the overall accuracy of the assignments between the lowest and highest levels of library completeness at the tribe and subfamily level. Conclusions Our results suggest that when using a strict tree-based criterion for higher taxon assignment with DNA barcodes, the likelihood of assigning a query a genus name incorrectly is very low, if a genus name is provided it has a high likelihood of being accurate, and if no genus match is available the query can nevertheless be assigned to a subfamily with high accuracy regardless of library completeness. DNA barcoding often correctly assigned sphingid moths to higher taxa when species matches were unavailable, suggesting that barcode reference libraries can be useful for higher taxon assignments long before they achieve complete species coverage. PMID:21806794

Moorea BIOCODE barcode library as a tool for understanding predator-prey interactions: insights into the diet of common predatory coral reef fishes

NASA Astrophysics Data System (ADS)

Leray, M.; Boehm, J. T.; Mills, S. C.; Meyer, C. P.

2012-06-01

Identifying species involved in consumer-resource interactions is one of the main limitations in the construction of food webs. DNA barcoding of prey items in predator guts provides a valuable tool for characterizing trophic interactions, but the method relies on the availability of reference sequences to which prey sequences can be matched. In this study, we demonstrate that the COI sequence library of the Moorea BIOCODE project, an ecosystem-level barcode initiative, enables the identification of a large proportion of semi-digested fish, crustacean and mollusks found in the guts of three Hawkfish and two Squirrelfish species. While most prey remains lacked diagnostic morphological characters, 94% of the prey found in 67 fishes had >98% sequence similarity with BIOCODE reference sequences. Using this species-level prey identification, we demonstrate how DNA barcoding can provide insights into resource partitioning, predator feeding behaviors and the consequences of predation on ecosystem function.

Opening the treasure chest: A DNA-barcoding primer set for most higher taxa of Central European birds and mammals from museum collections

PubMed Central

Schäffer, Sylvia; Zachos, Frank E.

2017-01-01

DNA-barcoding is a rapidly developing method for efficiently identifying samples to species level by means of short standard DNA sequences. However, reliable species assignment requires the availability of a comprehensive DNA barcode reference library, and hence numerous initiatives aim at generating such barcode databases for particular taxa or geographic regions. Historical museum collections represent a potentially invaluable source for the DNA-barcoding of many taxa. This is particularly true for birds and mammals, for which collecting fresh (voucher) material is often very difficult to (nearly) impossible due to the special animal welfare and conservation regulations that apply to vertebrates in general, and birds and mammals in particular. Moreover, even great efforts might not guarantee sufficiently complete sampling of fresh material in a short period of time. DNA extracted from historical samples is usually degraded, such that only short fragments can be amplified, rendering the recovery of the barcoding region as a single fragment impossible. Here, we present a new set of primers that allows the efficient amplification and sequencing of the entire barcoding region in most higher taxa of Central European birds and mammals in six overlapping fragments, thus greatly increasing the value of historical museum collections for generating DNA barcode reference libraries. Applying our new primer set in recently established NGS protocols promises to further increase the efficiency of barcoding old bird and mammal specimens. PMID:28358863

Opening the treasure chest: A DNA-barcoding primer set for most higher taxa of Central European birds and mammals from museum collections.

PubMed

Schäffer, Sylvia; Zachos, Frank E; Koblmüller, Stephan

2017-01-01

DNA-barcoding is a rapidly developing method for efficiently identifying samples to species level by means of short standard DNA sequences. However, reliable species assignment requires the availability of a comprehensive DNA barcode reference library, and hence numerous initiatives aim at generating such barcode databases for particular taxa or geographic regions. Historical museum collections represent a potentially invaluable source for the DNA-barcoding of many taxa. This is particularly true for birds and mammals, for which collecting fresh (voucher) material is often very difficult to (nearly) impossible due to the special animal welfare and conservation regulations that apply to vertebrates in general, and birds and mammals in particular. Moreover, even great efforts might not guarantee sufficiently complete sampling of fresh material in a short period of time. DNA extracted from historical samples is usually degraded, such that only short fragments can be amplified, rendering the recovery of the barcoding region as a single fragment impossible. Here, we present a new set of primers that allows the efficient amplification and sequencing of the entire barcoding region in most higher taxa of Central European birds and mammals in six overlapping fragments, thus greatly increasing the value of historical museum collections for generating DNA barcode reference libraries. Applying our new primer set in recently established NGS protocols promises to further increase the efficiency of barcoding old bird and mammal specimens.

A reliable DNA barcode reference library for the identification of the North European shelf fish fauna.

PubMed

Knebelsberger, Thomas; Landi, Monica; Neumann, Hermann; Kloppmann, Matthias; Sell, Anne F; Campbell, Patrick D; Laakmann, Silke; Raupach, Michael J; Carvalho, Gary R; Costa, Filipe O

2014-09-01

Valid fish species identification is an essential step both for fundamental science and fisheries management. The traditional identification is mainly based on external morphological diagnostic characters, leading to inconsistent results in many cases. Here, we provide a sequence reference library based on mitochondrial cytochrome c oxidase subunit I (COI) for a valid identification of 93 North Atlantic fish species originating from the North Sea and adjacent waters, including many commercially exploited species. Neighbour-joining analysis based on K2P genetic distances formed nonoverlapping clusters for all species with a ≥99% bootstrap support each. Identification was successful for 100% of the species as the minimum genetic distance to the nearest neighbour always exceeded the maximum intraspecific distance. A barcoding gap was apparent for the whole data set. Within-species distances ranged from 0 to 2.35%, while interspecific distances varied between 3.15 and 28.09%. Distances between congeners were on average 51-fold higher than those within species. The validation of the sequence library by applying BOLDs barcode index number (BIN) analysis tool and a ranking system demonstrated high taxonomic reliability of the DNA barcodes for 85% of the investigated fish species. Thus, the sequence library presented here can be confidently used as a benchmark for identification of at least two-thirds of the typical fish species recorded for the North Sea. © 2014 John Wiley & Sons Ltd.

Barcoding and Border Biosecurity: Identifying Cyprinid Fishes in the Aquarium Trade

PubMed Central

Collins, Rupert A.; Armstrong, Karen F.; Meier, Rudolf; Yi, Youguang; Brown, Samuel D. J.; Cruickshank, Robert H.; Keeling, Suzanne; Johnston, Colin

2012-01-01

Background Poorly regulated international trade in ornamental fishes poses risks to both biodiversity and economic activity via invasive alien species and exotic pathogens. Border security officials need robust tools to confirm identifications, often requiring hard-to-obtain taxonomic literature and expertise. DNA barcoding offers a potentially attractive tool for quarantine inspection, but has yet to be scrutinised for aquarium fishes. Here, we present a barcoding approach for ornamental cyprinid fishes by: (1) expanding current barcode reference libraries; (2) assessing barcode congruence with morphological identifications under numerous scenarios (e.g. inclusion of GenBank data, presence of singleton species, choice of analytical method); and (3) providing supplementary information to identify difficult species. Methodology/Principal Findings We sampled 172 ornamental cyprinid fish species from the international trade, and provide data for 91 species currently unrepresented in reference libraries (GenBank/Bold). DNA barcodes were found to be highly congruent with our morphological assignments, achieving success rates of 90–99%, depending on the method used (neighbour-joining monophyly, bootstrap, nearest neighbour, GMYC, percent threshold). Inclusion of data from GenBank (additional 157 spp.) resulted in a more comprehensive library, but at a cost to success rate due to the increased number of singleton species. In addition to DNA barcodes, our study also provides supporting data in the form of specimen images, morphological characters, taxonomic bibliography, preserved vouchers, and nuclear rhodopsin sequences. Using this nuclear rhodopsin data we also uncovered evidence of interspecific hybridisation, and highlighted unrecognised diversity within popular aquarium species, including the endangered Indian barb Puntius denisonii. Conclusions/Significance We demonstrate that DNA barcoding provides a highly effective biosecurity tool for rapidly identifying ornamental fishes. In cases where DNA barcodes are unable to offer an identification, we improve on previous studies by consolidating supplementary information from multiple data sources, and empower biosecurity agencies to confidently identify high-risk fishes in the aquarium trade. PMID:22276096

Large-scale DNA Barcode Library Generation for Biomolecule Identification in High-throughput Screens.

PubMed

Lyons, Eli; Sheridan, Paul; Tremmel, Georg; Miyano, Satoru; Sugano, Sumio

2017-10-24

High-throughput screens allow for the identification of specific biomolecules with characteristics of interest. In barcoded screens, DNA barcodes are linked to target biomolecules in a manner allowing for the target molecules making up a library to be identified by sequencing the DNA barcodes using Next Generation Sequencing. To be useful in experimental settings, the DNA barcodes in a library must satisfy certain constraints related to GC content, homopolymer length, Hamming distance, and blacklisted subsequences. Here we report a novel framework to quickly generate large-scale libraries of DNA barcodes for use in high-throughput screens. We show that our framework dramatically reduces the computation time required to generate large-scale DNA barcode libraries, compared with a naїve approach to DNA barcode library generation. As a proof of concept, we demonstrate that our framework is able to generate a library consisting of one million DNA barcodes for use in a fragment antibody phage display screening experiment. We also report generating a general purpose one billion DNA barcode library, the largest such library yet reported in literature. Our results demonstrate the value of our novel large-scale DNA barcode library generation framework for use in high-throughput screening applications.

Filling reference gaps via assembling DNA barcodes using high-throughput sequencing—moving toward barcoding the world

PubMed Central

Zhou, Chengran

2017-01-01

Abstract Over the past decade, biodiversity researchers have dedicated tremendous efforts to constructing DNA reference barcodes for rapid species registration and identification. Although analytical cost for standard DNA barcoding has been significantly reduced since early 2000, further dramatic reduction in barcoding costs is unlikely because Sanger sequencing is approaching its limits in throughput and chemistry cost. Constraints in barcoding cost not only led to unbalanced barcoding efforts around the globe, but also prevented high-throughput sequencing (HTS)–based taxonomic identification from applying binomial species names, which provide crucial linkages to biological knowledge. We developed an Illumina-based pipeline, HIFI-Barcode, to produce full-length Cytochrome c oxidase subunit I (COI) barcodes from pooled polymerase chain reaction amplicons generated by individual specimens. The new pipeline generated accurate barcode sequences that were comparable to Sanger standards, even for different haplotypes of the same species that were only a few nucleotides different from each other. Additionally, the new pipeline was much more sensitive in recovering amplicons at low quantity. The HIFI-Barcode pipeline successfully recovered barcodes from more than 78% of the polymerase chain reactions that didn’t show clear bands on the electrophoresis gel. Moreover, sequencing results based on the single molecular sequencing platform Pacbio confirmed the accuracy of the HIFI-Barcode results. Altogether, the new pipeline can provide an improved solution to produce full-length reference barcodes at about one-tenth of the current cost, enabling construction of comprehensive barcode libraries for local fauna, leading to a feasible direction for DNA barcoding global biomes. PMID:29077841

Filling reference gaps via assembling DNA barcodes using high-throughput sequencing-moving toward barcoding the world.

PubMed

Liu, Shanlin; Yang, Chentao; Zhou, Chengran; Zhou, Xin

2017-12-01

Over the past decade, biodiversity researchers have dedicated tremendous efforts to constructing DNA reference barcodes for rapid species registration and identification. Although analytical cost for standard DNA barcoding has been significantly reduced since early 2000, further dramatic reduction in barcoding costs is unlikely because Sanger sequencing is approaching its limits in throughput and chemistry cost. Constraints in barcoding cost not only led to unbalanced barcoding efforts around the globe, but also prevented high-throughput sequencing (HTS)-based taxonomic identification from applying binomial species names, which provide crucial linkages to biological knowledge. We developed an Illumina-based pipeline, HIFI-Barcode, to produce full-length Cytochrome c oxidase subunit I (COI) barcodes from pooled polymerase chain reaction amplicons generated by individual specimens. The new pipeline generated accurate barcode sequences that were comparable to Sanger standards, even for different haplotypes of the same species that were only a few nucleotides different from each other. Additionally, the new pipeline was much more sensitive in recovering amplicons at low quantity. The HIFI-Barcode pipeline successfully recovered barcodes from more than 78% of the polymerase chain reactions that didn't show clear bands on the electrophoresis gel. Moreover, sequencing results based on the single molecular sequencing platform Pacbio confirmed the accuracy of the HIFI-Barcode results. Altogether, the new pipeline can provide an improved solution to produce full-length reference barcodes at about one-tenth of the current cost, enabling construction of comprehensive barcode libraries for local fauna, leading to a feasible direction for DNA barcoding global biomes. © The Authors 2017. Published by Oxford University Press.

Testing DNA barcode performance in 1000 species of European lepidoptera: large geographic distances have small genetic impacts.

PubMed

Huemer, Peter; Mutanen, Marko; Sefc, Kristina M; Hebert, Paul D N

2014-01-01

This study examines the performance of DNA barcodes (mt cytochrome c oxidase 1 gene) in the identification of 1004 species of Lepidoptera shared by two localities (Finland, Austria) that are 1600 km apart. Maximum intraspecific distances for the pooled data were less than 2% for 880 species (87.6%), while deeper divergence was detected in 124 species. Despite such variation, the overall DNA barcode library possessed diagnostic COI sequences for 98.8% of the taxa. Because a reference library based on Finnish specimens was highly effective in identifying specimens from Austria, we conclude that barcode libraries based on regional sampling can often be effective for a much larger area. Moreover, dispersal ability (poor, good) and distribution patterns (disjunct, fragmented, continuous, migratory) had little impact on levels of intraspecific geographic divergence. Furthermore, the present study revealed that, despite the intensity of past taxonomic work on European Lepidoptera, nearly 20% of the species shared by Austria and Finland require further work to clarify their status. Particularly discordant BIN (Barcode Index Number) cases should be checked to ascertain possible explanatory factors such as incorrect taxonomy, hybridization, introgression, and Wolbachia infections.

Assessing DNA Barcodes for Species Identification in North American Reptiles and Amphibians in Natural History Collections.

PubMed

Chambers, E Anne; Hebert, Paul D N

2016-01-01

High rates of species discovery and loss have led to the urgent need for more rapid assessment of species diversity in the herpetofauna. DNA barcoding allows for the preliminary identification of species based on sequence divergence. Prior DNA barcoding work on reptiles and amphibians has revealed higher biodiversity counts than previously estimated due to cases of cryptic and undiscovered species. Past studies have provided DNA barcodes for just 14% of the North American herpetofauna, revealing the need for expanded coverage. This study extends the DNA barcode reference library for North American herpetofauna, assesses the utility of this approach in aiding species delimitation, and examines the correspondence between current species boundaries and sequence clusters designated by the BIN system. Sequences were obtained from 730 specimens, representing 274 species (43%) from the North American herpetofauna. Mean intraspecific divergences were 1% and 3%, while average congeneric sequence divergences were 16% and 14% in amphibians and reptiles, respectively. BIN assignments corresponded with current species boundaries in 79% of amphibians, 100% of turtles, and 60% of squamates. Deep divergences (>2%) were noted in 35% of squamate and 16% of amphibian species, and low divergences (<2%) occurred in 12% of reptiles and 23% of amphibians, patterns reflected in BIN assignments. Sequence recovery declined with specimen age, and variation in recovery success was noted among collections. Within collections, barcodes effectively flagged seven mislabeled tissues, and barcode fragments were recovered from five formalin-fixed specimens. This study demonstrates that DNA barcodes can effectively flag errors in museum collections, while BIN splits and merges reveal taxa belonging to deeply diverged or hybridizing lineages. This study is the first effort to compile a reference library of DNA barcodes for herpetofauna on a continental scale.

Assessing DNA Barcodes for Species Identification in North American Reptiles and Amphibians in Natural History Collections

PubMed Central

Chambers, E. Anne; Hebert, Paul D. N.

2016-01-01

Background High rates of species discovery and loss have led to the urgent need for more rapid assessment of species diversity in the herpetofauna. DNA barcoding allows for the preliminary identification of species based on sequence divergence. Prior DNA barcoding work on reptiles and amphibians has revealed higher biodiversity counts than previously estimated due to cases of cryptic and undiscovered species. Past studies have provided DNA barcodes for just 14% of the North American herpetofauna, revealing the need for expanded coverage. Methodology/Principal Findings This study extends the DNA barcode reference library for North American herpetofauna, assesses the utility of this approach in aiding species delimitation, and examines the correspondence between current species boundaries and sequence clusters designated by the BIN system. Sequences were obtained from 730 specimens, representing 274 species (43%) from the North American herpetofauna. Mean intraspecific divergences were 1% and 3%, while average congeneric sequence divergences were 16% and 14% in amphibians and reptiles, respectively. BIN assignments corresponded with current species boundaries in 79% of amphibians, 100% of turtles, and 60% of squamates. Deep divergences (>2%) were noted in 35% of squamate and 16% of amphibian species, and low divergences (<2%) occurred in 12% of reptiles and 23% of amphibians, patterns reflected in BIN assignments. Sequence recovery declined with specimen age, and variation in recovery success was noted among collections. Within collections, barcodes effectively flagged seven mislabeled tissues, and barcode fragments were recovered from five formalin-fixed specimens. Conclusions/Significance This study demonstrates that DNA barcodes can effectively flag errors in museum collections, while BIN splits and merges reveal taxa belonging to deeply diverged or hybridizing lineages. This study is the first effort to compile a reference library of DNA barcodes for herpetofauna on a continental scale. PMID:27116180

A DNA barcode library for ground beetles of Germany: the genus Amara Bonelli, 1810 (Insecta, Coleoptera, Carabidae)

PubMed Central

Raupach, Michael J.; Hannig, Karsten; Moriniére, Jérôme; Hendrich, Lars

2018-01-01

Abstract The genus Amara Bonelli, 1810 is a very speciose and taxonomically difficult genus of the Carabidae. The identification of many of the species is accomplished with considerable difficulty, in particular for females and immature stages. In this study the effectiveness of DNA barcoding, the most popular method for molecular species identification, was examined to discriminate various species of this genus from Central Europe. DNA barcodes from 690 individuals and 47 species were analysed, including sequences from previous studies and more than 350 newly generated DNA barcodes. Our analysis revealed unique BINs for 38 species (81%). Interspecific K2P distances below 2.2% were found for three species pairs and one species trio, including haplotype sharing between Amara alpina/Amara torrida and Amara communis/Amara convexior/Amara makolskii. This study represents another step in generating an extensive reference library of DNA barcodes for carabids, highly valuable bioindicators for characterizing disturbances in various habitats. PMID:29853775

Improved protocols to accelerate the assembly of DNA barcode reference libraries for freshwater zooplankton.

PubMed

Elías-Gutiérrez, Manuel; Valdez-Moreno, Martha; Topan, Janet; Young, Monica R; Cohuo-Colli, José Angel

2018-03-01

Currently, freshwater zooplankton sampling and identification methodologies have remained virtually unchanged since they were first established in the beginning of the XX century. One major contributing factor to this slow progress is the limited success of modern genetic methodologies, such as DNA barcoding, in several of the main groups. This study demonstrates improved protocols which enable the rapid assessment of most animal taxa inhabiting any freshwater system by combining the use of light traps, careful fixation at low temperatures using ethanol, and zooplankton-specific primers. We DNA-barcoded 2,136 specimens from a diverse array of taxonomic assemblages (rotifers, mollusks, mites, crustaceans, insects, and fishes) from several Canadian and Mexican lakes with an average sequence success rate of 85.3%. In total, 325 Barcode Index Numbers (BINs) were detected with only three BINs (two cladocerans and one copepod) shared between Canada and Mexico, suggesting a much narrower distribution range of freshwater zooplankton than previously thought. This study is the first to broadly explore the metazoan biodiversity of freshwater systems with DNA barcodes to construct a reference library that represents the first step for future programs which aim to monitor ecosystem health, track invasive species, or improve knowledge of the ecology and distribution of freshwater zooplankton.

The campaign to DNA barcode all fishes, FISH-BOL.

PubMed

Ward, R D; Hanner, R; Hebert, P D N

2009-02-01

FISH-BOL, the Fish Barcode of Life campaign, is an international research collaboration that is assembling a standardized reference DNA sequence library for all fishes. Analysis is targeting a 648 base pair region of the mitochondrial cytochrome c oxidase I (COI) gene. More than 5000 species have already been DNA barcoded, with an average of five specimens per species, typically vouchers with authoritative identifications. The barcode sequence from any fish, fillet, fin, egg or larva can be matched against these reference sequences using BOLD; the Barcode of Life Data System (http://www.barcodinglife.org). The benefits of barcoding fishes include facilitating species identification, highlighting cases of range expansion for known species, flagging previously overlooked species and enabling identifications where traditional methods cannot be applied. Results thus far indicate that barcodes separate c. 98 and 93% of already described marine and freshwater fish species, respectively. Several specimens with divergent barcode sequences have been confirmed by integrative taxonomic analysis as new species. Past concerns in relation to the use of fish barcoding for species discrimination are discussed. These include hybridization, recent radiations, regional differentiation in barcode sequences and nuclear copies of the barcode region. However, current results indicate these issues are of little concern for the great majority of specimens.

A DNA Barcode Library for North American Pyraustinae (Lepidoptera: Pyraloidea: Crambidae).

PubMed

Yang, Zhaofu; Landry, Jean-François; Hebert, Paul D N

2016-01-01

Although members of the crambid subfamily Pyraustinae are frequently important crop pests, their identification is often difficult because many species lack conspicuous diagnostic morphological characters. DNA barcoding employs sequence diversity in a short standardized gene region to facilitate specimen identifications and species discovery. This study provides a DNA barcode reference library for North American pyraustines based upon the analysis of 1589 sequences recovered from 137 nominal species, 87% of the fauna. Data from 125 species were barcode compliant (>500bp, <1% n), and 99 of these taxa formed a distinct cluster that was assigned to a single BIN. The other 26 species were assigned to 56 BINs, reflecting frequent cases of deep intraspecific sequence divergence and a few instances of barcode sharing, creating a total of 155 BINs. Two systems for OTU designation, ABGD and BIN, were examined to check the correspondence between current taxonomy and sequence clusters. The BIN system performed better than ABGD in delimiting closely related species, while OTU counts with ABGD were influenced by the value employed for relative gap width. Different species with low or no interspecific divergence may represent cases of unrecognized synonymy, whereas those with high intraspecific divergence require further taxonomic scrutiny as they may involve cryptic diversity. The barcode library developed in this study will also help to advance understanding of relationships among species of Pyraustinae.

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

PubMed

Nithaniyal, Stalin; Vassou, Sophie Lorraine; Poovitha, Sundar; Raju, Balaji; Parani, Madasamy

2017-02-01

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.

DNA Barcoding Identifies Argentine Fishes from Marine and Brackish Waters

PubMed Central

Mabragaña, Ezequiel; Díaz de Astarloa, Juan Martín; Hanner, Robert; Zhang, Junbin; González Castro, Mariano

2011-01-01

Background DNA barcoding has been advanced as a promising tool to aid species identification and discovery through the use of short, standardized gene targets. Despite extensive taxonomic studies, for a variety of reasons the identification of fishes can be problematic, even for experts. DNA barcoding is proving to be a useful tool in this context. However, its broad application is impeded by the need to construct a comprehensive reference sequence library for all fish species. Here, we make a regional contribution to this grand challenge by calibrating the species discrimination efficiency of barcoding among 125 Argentine fish species, representing nearly one third of the known fauna, and examine the utility of these data to address several key taxonomic uncertainties pertaining to species in this region. Methodology/Principal Findings Specimens were collected and morphologically identified during crusies conducted between 2005 and 2008. The standard BARCODE fragment of COI was amplified and bi-directionally sequenced from 577 specimens (mean of 5 specimens/species), and all specimens and sequence data were archived and interrogated using analytical tools available on the Barcode of Life Data System (BOLD; www.barcodinglife.org). Nearly all species exhibited discrete clusters of closely related haplogroups which permitted the discrimination of 95% of the species (i.e. 119/125) examined while cases of shared haplotypes were detected among just three species-pairs. Notably, barcoding aided the identification of a new species of skate, Dipturus argentinensis, permitted the recognition of Genypterus brasiliensis as a valid species and questions the generic assignment of Paralichthys isosceles. Conclusions/Significance This study constitutes a significant contribution to the global barcode reference sequence library for fishes and demonstrates the utility of barcoding for regional species identification. As an independent assessment of alpha taxonomy, barcodes provide robust support for most morphologically based taxon concepts and also highlight key areas of taxonomic uncertainty worthy of reappraisal. PMID:22174860

First DNA Barcode Reference Library for the Identification of South American Freshwater Fish from the Lower Paraná River

PubMed Central

Brancolini, Florencia; del Pazo, Felipe; Posner, Victoria Maria; Grimberg, Alexis; Arranz, Silvia Eda

2016-01-01

Valid fish species identification is essential for biodiversity conservation and fisheries management. Here, we provide a sequence reference library based on mitochondrial cytochrome c oxidase subunit I for a valid identification of 79 freshwater fish species from the Lower Paraná River. Neighbour-joining analysis based on K2P genetic distances formed non-overlapping clusters for almost all species with a ≥99% bootstrap support each. Identification was successful for 97.8% of species as the minimum genetic distance to the nearest neighbour exceeded the maximum intraspecific distance in all these cases. A barcoding gap of 2.5% was apparent for the whole data set with the exception of four cases. Within-species distances ranged from 0.00% to 7.59%, while interspecific distances varied between 4.06% and 19.98%, without considering Odontesthes species with a minimum genetic distance of 0%. Sequence library validation was performed by applying BOLDs BIN analysis tool, Poisson Tree Processes model and Automatic Barcode Gap Discovery, along with a reliable taxonomic assignment by experts. Exhaustive revision of vouchers was performed when a conflicting assignment was detected after sequence analysis and BIN discordance evaluation. Thus, the sequence library presented here can be confidently used as a benchmark for identification of half of the fish species recorded for the Lower Paraná River. PMID:27442116

A DNA Barcode Library for North American Ephemeroptera: Progress and Prospects

PubMed Central

Webb, Jeffrey M.; Jacobus, Luke M.; Funk, David H.; Zhou, Xin; Kondratieff, Boris; Geraci, Christy J.; DeWalt, R. Edward; Baird, Donald J.; Richard, Barton; Phillips, Iain; Hebert, Paul D. N.

2012-01-01

DNA barcoding of aquatic macroinvertebrates holds much promise as a tool for taxonomic research and for providing the reliable identifications needed for water quality assessment programs. A prerequisite for identification using barcodes is a reliable reference library. We gathered 4165 sequences from the barcode region of the mitochondrial cytochrome c oxidase subunit I gene representing 264 nominal and 90 provisional species of mayflies (Insecta: Ephemeroptera) from Canada, Mexico, and the United States. No species shared barcode sequences and all can be identified with barcodes with the possible exception of some Caenis. Minimum interspecific distances ranged from 0.3–24.7% (mean: 12.5%), while the average intraspecific divergence was 1.97%. The latter value was inflated by the presence of very high divergences in some taxa. In fact, nearly 20% of the species included two or three haplotype clusters showing greater than 5.0% sequence divergence and some values are as high as 26.7%. Many of the species with high divergences are polyphyletic and likely represent species complexes. Indeed, many of these polyphyletic species have numerous synonyms and individuals in some barcode clusters show morphological attributes characteristic of the synonymized species. In light of our findings, it is imperative that type or topotype specimens be sequenced to correctly associate barcode clusters with morphological species concepts and to determine the status of currently synonymized species. PMID:22666447

Complete DNA barcode reference library for a country's butterfly fauna reveals high performance for temperate Europe

PubMed Central

Dincă, Vlad; Zakharov, Evgeny V.; Hebert, Paul D. N.; Vila, Roger

2011-01-01

DNA barcoding aims to accelerate species identification and discovery, but performance tests have shown marked differences in identification success. As a consequence, there remains a great need for comprehensive studies which objectively test the method in groups with a solid taxonomic framework. This study focuses on the 180 species of butterflies in Romania, accounting for about one third of the European butterfly fauna. This country includes five eco-regions, the highest of any in the European Union, and is a good representative for temperate areas. Morphology and DNA barcodes of more than 1300 specimens were carefully studied and compared. Our results indicate that 90 per cent of the species form barcode clusters allowing their reliable identification. The remaining cases involve nine closely related species pairs, some whose taxonomic status is controversial or that hybridize regularly. Interestingly, DNA barcoding was found to be the most effective identification tool, outperforming external morphology, and being slightly better than male genitalia. Romania is now the first country to have a comprehensive DNA barcode reference database for butterflies. Similar barcoding efforts based on comprehensive sampling of specific geographical regions can act as functional modules that will foster the early application of DNA barcoding while a global system is under development. PMID:20702462

A DNA barcode library for Germany's mayflies, stoneflies and caddisflies (Ephemeroptera, Plecoptera and Trichoptera).

PubMed

Morinière, Jérôme; Hendrich, Lars; Balke, Michael; Beermann, Arne J; König, Tobias; Hess, Monika; Koch, Stefan; Müller, Reinhard; Leese, Florian; Hebert, Paul D N; Hausmann, Axel; Schubart, Christoph D; Haszprunar, Gerhard

2017-11-01

Mayflies, stoneflies and caddisflies (Ephemeroptera, Plecoptera and Trichoptera) are prominent representatives of aquatic macroinvertebrates, commonly used as indicator organisms for water quality and ecosystem assessments. However, unambiguous morphological identification of EPT species, especially their immature life stages, is a challenging, yet fundamental task. A comprehensive DNA barcode library based upon taxonomically well-curated specimens is needed to overcome the problematic identification. Once available, this library will support the implementation of fast, cost-efficient and reliable DNA-based identifications and assessments of ecological status. This study represents a major step towards a DNA barcode reference library as it covers for two-thirds of Germany's EPT species including 2,613 individuals belonging to 363 identified species. As such, it provides coverage for 38 of 44 families (86%) and practically all major bioindicator species. DNA barcode compliant sequences (≥500 bp) were recovered from 98.74% of the analysed specimens. Whereas most species (325, i.e., 89.53%) were unambiguously assigned to a single Barcode Index Number (BIN) by its COI sequence, 38 species (18 Ephemeroptera, nine Plecoptera and 11 Trichoptera) were assigned to a total of 89 BINs. Most of these additional BINs formed nearest neighbour clusters, reflecting the discrimination of geographical subclades of a currently recognized species. BIN sharing was uncommon, involving only two species pairs of Ephemeroptera. Interestingly, both maximum pairwise and nearest neighbour distances were substantially higher for Ephemeroptera compared to Plecoptera and Trichoptera, possibly indicating older speciation events, stronger positive selection or faster rate of molecular evolution. © 2017 John Wiley & Sons Ltd.

DNA barcoding of vouchered xylarium wood specimens of nine endangered Dalbergia species.

PubMed

Yu, Min; Jiao, Lichao; Guo, Juan; Wiedenhoeft, Alex C; He, Tuo; Jiang, Xiaomei; Yin, Yafang

2017-12-01

ITS2+ trnH - psbA was the best combination of DNA barcode to resolve the Dalbergia wood species studied. We demonstrate the feasibility of building a DNA barcode reference database using xylarium wood specimens. The increase in illegal logging and timber trade of CITES-listed tropical species necessitates the development of unambiguous identification methods at the species level. For these methods to be fully functional and deployable for law enforcement, they must work using wood or wood products. DNA barcoding of wood has been promoted as a promising tool for species identification; however, the main barrier to extensive application of DNA barcoding to wood is the lack of a comprehensive and reliable DNA reference library of barcodes from wood. In this study, xylarium wood specimens of nine Dalbergia species were selected from the Wood Collection of the Chinese Academy of Forestry and DNA was then extracted from them for further PCR amplification of eight potential DNA barcode sequences (ITS2, matK, trnL, trnH-psbA, trnV-trnM1, trnV-trnM2, trnC-petN, and trnS-trnG). The barcodes were tested singly and in combination for species-level discrimination ability by tree-based [neighbor-joining (NJ)] and distance-based (TaxonDNA) methods. We found that the discrimination ability of DNA barcodes in combination was higher than any single DNA marker among the Dalbergia species studied, with the best two-marker combination of ITS2+trnH-psbA analyzed with NJ trees performing the best (100% accuracy). These barcodes are relatively short regions (<350 bp) and amplification reactions were performed with high success (≥90%) using wood as the source material, a necessary factor to apply DNA barcoding to timber trade. The present results demonstrate the feasibility of using vouchered xylarium specimens to build DNA barcoding reference databases.

Probing planetary biodiversity with DNA barcodes: The Noctuoidea of North America

PubMed Central

Lafontaine, J. Donald; Schmidt, B. Christian; deWaard, Jeremy R.; Zakharov, Evgeny V.; Hebert, Paul D. N.

2017-01-01

This study reports the assembly of a DNA barcode reference library for species in the lepidopteran superfamily Noctuoidea from Canada and the USA. Based on the analysis of 69,378 specimens, the library provides coverage for 97.3% of the noctuoid fauna (3565 of 3664 species). In addition to verifying the strong performance of DNA barcodes in the discrimination of these species, the results indicate close congruence between the number of species analyzed (3565) and the number of sequence clusters (3816) recognized by the Barcode Index Number (BIN) system. Distributional patterns across 12 North American ecoregions are examined for the 3251 species that have GPS data while BIN analysis is used to quantify overlap between the noctuoid faunas of North America and other zoogeographic regions. This analysis reveals that 90% of North American noctuoids are endemic and that just 7.5% and 1.8% of BINs are shared with the Neotropics and with the Palearctic, respectively. One third (29) of the latter species are recent introductions and, as expected, they possess low intraspecific divergences. PMID:28570635

Building a DNA barcode library of Alaska's non-marine arthropods.

PubMed

Sikes, Derek S; Bowser, Matthew; Morton, John M; Bickford, Casey; Meierotto, Sarah; Hildebrandt, Kyndall

2017-03-01

Climate change may result in ecological futures with novel species assemblages, trophic mismatch, and mass extinction. Alaska has a limited taxonomic workforce to address these changes. We are building a DNA barcode library to facilitate a metabarcoding approach to monitoring non-marine arthropods. Working with the Canadian Centre for DNA Barcoding, we obtained DNA barcodes from recently collected and authoritatively identified specimens in the University of Alaska Museum (UAM) Insect Collection and the Kenai National Wildlife Refuge collection. We submitted tissues from 4776 specimens, of which 81% yielded DNA barcodes representing 1662 species and 1788 Barcode Index Numbers (BINs), of primarily terrestrial, large-bodied arthropods. This represents 84% of the species available for DNA barcoding in the UAM Insect Collection. There are now 4020 Alaskan arthropod species represented by DNA barcodes, after including all records in Barcode of Life Data Systems (BOLD) of species that occur in Alaska - i.e., 48.5% of the 8277 Alaskan, non-marine-arthropod, named species have associated DNA barcodes. An assessment of the identification power of the library in its current state yielded fewer species-level identifications than expected, but the results were not discouraging. We believe we are the first to deliberately begin development of a DNA barcode library of the entire arthropod fauna for a North American state or province. Although far from complete, this library will become increasingly valuable as more species are added and costs to obtain DNA sequences fall.

A DNA Barcode Library for Korean Chironomidae (Insecta: Diptera) and Indexes for Defining Barcode Gap

PubMed Central

Kim, Sungmin; Song, Kyo-Hong; Ree, Han-Il; Kim, Won

2012-01-01

Non-biting midges (Diptera: Chironomidae) are a diverse population that commonly causes respiratory allergies in humans. Chironomid larvae can be used to indicate freshwater pollution, but accurate identification on the basis of morphological characteristics is difficult. In this study, we constructed a mitochondrial cytochrome c oxidase subunit I (COI)-based DNA barcode library for Korean chironomids. This library consists of 211 specimens from 49 species, including adults and unidentified larvae. The interspecies and intraspecies COI sequence variations were analyzed. Sophisticated indexes were developed in order to properly evaluate indistinct barcode gaps that are created by insufficient sampling on both the interspecies and intraspecies levels and by variable mutation rates across taxa. In a variety of insect datasets, these indexes were useful for re-evaluating large barcode datasets and for defining COI barcode gaps. The COI-based DNA barcode library will provide a rapid and reliable tool for the molecular identification of Korean chironomid species. Furthermore, this reverse-taxonomic approach will be improved by the continuous addition of other speceis’ sequences to the library. PMID:22138764

Untangling taxonomy: a DNA barcode reference library for Canadian spiders.

PubMed

Blagoev, Gergin A; deWaard, Jeremy R; Ratnasingham, Sujeevan; deWaard, Stephanie L; Lu, Liuqiong; Robertson, James; Telfer, Angela C; Hebert, Paul D N

2016-01-01

Approximately 1460 species of spiders have been reported from Canada, 3% of the global fauna. This study provides a DNA barcode reference library for 1018 of these species based upon the analysis of more than 30,000 specimens. The sequence results show a clear barcode gap in most cases with a mean intraspecific divergence of 0.78% vs. a minimum nearest-neighbour (NN) distance averaging 7.85%. The sequences were assigned to 1359 Barcode index numbers (BINs) with 1344 of these BINs composed of specimens belonging to a single currently recognized species. There was a perfect correspondence between BIN membership and a known species in 795 cases, while another 197 species were assigned to two or more BINs (556 in total). A few other species (26) were involved in BIN merges or in a combination of merges and splits. There was only a weak relationship between the number of specimens analysed for a species and its BIN count. However, three species were clear outliers with their specimens being placed in 11-22 BINs. Although all BIN splits need further study to clarify the taxonomic status of the entities involved, DNA barcodes discriminated 98% of the 1018 species. The present survey conservatively revealed 16 species new to science, 52 species new to Canada and major range extensions for 426 species. However, if most BIN splits detected in this study reflect cryptic taxa, the true species count for Canadian spiders could be 30-50% higher than currently recognized. © 2015 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd.

Establishment of a standard reference material (SRM) herbal DNA barcode library of Vitex negundo L. (lagundi) for quality control measures.

PubMed

Olivar, Jay Edneil C; Alaba, Joanner Paulus Erik P; Atienza, Jose Francisco M; Tan, Jerick Jeffrey S; Umali, Maximo T; Alejandro, Grecebio Jonathan D

2016-05-01

The majority of the population in the Philippines relies on herbal products as their primary source for their healthcare needs. After the recognition of Vitex negundo L. (lagundi) as an important and effective alternative medicine for cough, sore throat, asthma and fever by the Philippine Department of Health (DOH), there was an increase in the production of lagundi-based herbal products in the form of teas, capsules and syrups. The efficiency of these products is greatly reliant on the use of authentic plant material, and to this day no standard protocol has been established to authenticate plant materials. DNA barcoding offers a quick and reliable species authentication tool, but its application to plant material has been less successful due to (1) lack of a standard DNA barcoding loci in plants and (2) poor DNA yield from powderised plant products. This study reports the successful application of DNA barcoding in the authentication of five V. negundo herbal products sold in the Philippines. Also, the first standard reference material (SRM) herbal library for the recognition of authentic V. negundo samples was established using 42 gene accessions of ITS, psbA-trnH and matK barcoding loci. Authentication of the herbal products utilised the SRM following the BLASTn and maximum-likelihood (ML) tree construction criterion. Barcode sequences were retrieved for ITS and psbA-trnH of all products tested and the results of the study revealed that only one out of five herbal products satisfied both BLASTn and ML criterion and was considered to contain authentic V. negundo. The results prompt the urgent need to utilise DNA barcoding in authenticating herbal products available in the Philippine market. Authentication of these products will secure consumer health by preventing the negative effects of adulteration, substitution and contamination.

A comprehensive DNA barcode database for Central European beetles with a focus on Germany: adding more than 3500 identified species to BOLD.

PubMed

Hendrich, Lars; Morinière, Jérôme; Haszprunar, Gerhard; Hebert, Paul D N; Hausmann, Axel; Köhler, Frank; Balke, Michael

2015-07-01

Beetles are the most diverse group of animals and are crucial for ecosystem functioning. In many countries, they are well established for environmental impact assessment, but even in the well-studied Central European fauna, species identification can be very difficult. A comprehensive and taxonomically well-curated DNA barcode library could remedy this deficit and could also link hundreds of years of traditional knowledge with next generation sequencing technology. However, such a beetle library is missing to date. This study provides the globally largest DNA barcode reference library for Coleoptera for 15 948 individuals belonging to 3514 well-identified species (53% of the German fauna) with representatives from 97 of 103 families (94%). This study is the first comprehensive regional test of the efficiency of DNA barcoding for beetles with a focus on Germany. Sequences ≥500 bp were recovered from 63% of the specimens analysed (15 948 of 25 294) with short sequences from another 997 specimens. Whereas most specimens (92.2%) could be unambiguously assigned to a single known species by sequence diversity at CO1, 1089 specimens (6.8%) were assigned to more than one Barcode Index Number (BIN), creating 395 BINs which need further study to ascertain if they represent cryptic species, mitochondrial introgression, or simply regional variation in widespread species. We found 409 specimens (2.6%) that shared a BIN assignment with another species, most involving a pair of closely allied species as 43 BINs were involved. Most of these taxa were separated by barcodes although sequence divergences were low. Only 155 specimens (0.97%) show identical or overlapping clusters. © 2014 John Wiley & Sons Ltd.

DNA Barcode Authentication of Saw Palmetto Herbal Dietary Supplements

PubMed Central

Little, Damon P.; Jeanson, Marc L.

2013-01-01

Herbal dietary supplements made from saw palmetto (Serenoa repens; Arecaceae) fruit are commonly consumed to ameliorate benign prostate hyperplasia. A novel DNA mini–barcode assay to accurately identify [specificity = 1.00 (95% confidence interval = 0.74–1.00); sensitivity = 1.00 (95% confidence interval = 0.66–1.00); n = 31] saw palmetto dietary supplements was designed from a DNA barcode reference library created for this purpose. The mini–barcodes were used to estimate the frequency of mislabeled saw palmetto herbal dietary supplements on the market in the United States of America. Of the 37 supplements examined, amplifiable DNA could be extracted from 34 (92%). Mini–barcode analysis of these supplements demonstrated that 29 (85%) contain saw palmetto and that 2 (6%) supplements contain related species that cannot be legally sold as herbal dietary supplements in the United States of America. The identity of 3 (9%) supplements could not be conclusively determined. PMID:24343362

DNA barcode authentication of saw palmetto herbal dietary supplements.

PubMed

Little, Damon P; Jeanson, Marc L

2013-12-17

Herbal dietary supplements made from saw palmetto (Serenoa repens; Arecaceae) fruit are commonly consumed to ameliorate benign prostate hyperplasia. A novel DNA mini-barcode assay to accurately identify [specificity = 1.00 (95% confidence interval = 0.74-1.00); sensitivity = 1.00 (95% confidence interval = 0.66-1.00); n = 31] saw palmetto dietary supplements was designed from a DNA barcode reference library created for this purpose. The mini-barcodes were used to estimate the frequency of mislabeled saw palmetto herbal dietary supplements on the market in the United States of America. Of the 37 supplements examined, amplifiable DNA could be extracted from 34 (92%). Mini-barcode analysis of these supplements demonstrated that 29 (85%) contain saw palmetto and that 2 (6%) supplements contain related species that cannot be legally sold as herbal dietary supplements in the United States of America. The identity of 3 (9%) supplements could not be conclusively determined.

QR Codes in the Library: "It's Not Your Mother's Barcode!"

ERIC Educational Resources Information Center

Dobbs, Cheri

2011-01-01

Barcode scanning has become more than just fun. Now libraries and businesses are leveraging barcode technology as an innovative tool to market their products and ideas. Developed and popularized in Japan, these Quick Response (QR) or two-dimensional barcodes allow marketers to provide interactive content in an otherwise static environment. In this…

Highlights of DNA Barcoding in identification of salient microorganisms like fungi.

PubMed

Dulla, E L; Kathera, C; Gurijala, H K; Mallakuntla, T R; Srinivasan, P; Prasad, V; Mopati, R D; Jasti, P K

2016-12-01

Fungi, the second largest kingdom of eukaryotic life, are diverse and widespread. Fungi play a distinctive role in the production of different products on industrial scale, like fungal enzymes, antibiotics, fermented foods, etc., to give storage stability and improved health to meet major global challenges. To utilize algae perfectly for human needs, and to pave the way for getting a healthy relationship with fungi, it is important to identify them in a quick and robust manner with molecular-based identification system. So, there is a technique that aims to provide a well-organized method for species level identifications and to contribute powerfully to taxonomic and biodiversity research is DNA Barcoding. DNA Barcoding is generally achieved by the retrieval of a short DNA sequence - the 'barcode' - from a standard part of the genome and that barcode is then compared with a library of reference barcode sequences derived from individuals of known identity for identification. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Generation and analysis of a barcode-tagged insertion mutant library in the fission yeast Schizosaccharomyces pombe.

PubMed

Chen, Bo-Ruei; Hale, Devin C; Ciolek, Peter J; Runge, Kurt W

2012-05-03

Barcodes are unique DNA sequence tags that can be used to specifically label individual mutants. The barcode-tagged open reading frame (ORF) haploid deletion mutant collections in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe allow for high-throughput mutant phenotyping because the relative growth of mutants in a population can be determined by monitoring the proportions of their associated barcodes. While these mutant collections have greatly facilitated genome-wide studies, mutations in essential genes are not present, and the roles of these genes are not as easily studied. To further support genome-scale research in S. pombe, we generated a barcode-tagged fission yeast insertion mutant library that has the potential of generating viable mutations in both essential and non-essential genes and can be easily analyzed using standard molecular biological techniques. An insertion vector containing a selectable ura4+ marker and a random barcode was used to generate a collection of 10,000 fission yeast insertion mutants stored individually in 384-well plates and as six pools of mixed mutants. Individual barcodes are flanked by Sfi I recognition sites and can be oligomerized in a unique orientation to facilitate barcode sequencing. Independent genetic screens on a subset of mutants suggest that this library contains a diverse collection of single insertion mutations. We present several approaches to determine insertion sites. This collection of S. pombe barcode-tagged insertion mutants is well-suited for genome-wide studies. Because insertion mutations may eliminate, reduce or alter the function of essential and non-essential genes, this library will contain strains with a wide range of phenotypes that can be assayed by their associated barcodes. The design of the barcodes in this library allows for barcode sequencing using next generation or standard benchtop cloning approaches.

Preparation and infrared/raman classification of 630 spectroscopically encoded styrene copolymers.

PubMed

Fenniri, Hicham; Chun, Sangki; Terreau, Owen; Bravo-Vasquez, Juan-Pablo

2008-01-01

The barcoded resins (BCRs) were introduced recently as a platform for encoded combinatorial chemistry. One of the main challenges yet to be overcome is the demonstration that a large number of BCRs could be generated and classified with high confidence. Here, we describe the synthesis and classification of 630 polystyrene-based copolymers prepared from the combinatorial association of 15 spectroscopically active styrene monomers. Each of the 630 copolymers displayed a unique vibrational fingerprint (infrared and Raman), which was converted into a spectral vector. To each of the 630 copolymers, a vector of the known (reference) composition was assigned. Unknown (prediction) vectors were decoded using multivariate data analysis. From the inner product of the reference and prediction vectors, a correlation map comparing 396 900 copolymer pairs (630 x 630) was generated. In 100% of the cases, the highest correlation was obtained for polymer pairs in which the reference and prediction vectors correspond to copolymers prepared from identical styrene monomers, thus demonstrating the high reliability of this encoding strategy. We have also established that the spectroscopic barcodes generated from the Raman and infrared spectra are independent of the copolymers' morphology (beaded versus bulk polymers). Besides the demonstration of the generality of the polymer barcoding strategy, the analytical methods developed here could in principle be extended to the investigation of the composition and purity of any other synthetic polymer and biopolymer library, or even scaffold-based combinatorial libraries.

Interbreeding among deeply divergent mitochondrial lineages in the American cockroach (Periplaneta americana)

NASA Astrophysics Data System (ADS)

von Beeren, Christoph; Stoeckle, Mark Y.; Xia, Joyce; Burke, Griffin; Kronauer, Daniel J. C.

2015-02-01

DNA barcoding promises to be a useful tool to identify pest species assuming adequate representation of genetic variants in a reference library. Here we examined mitochondrial DNA barcodes in a global urban pest, the American cockroach (Periplaneta americana). Our sampling effort generated 284 cockroach specimens, most from New York City, plus 15 additional U.S. states and six other countries, enabling the first large-scale survey of P. americana barcode variation. Periplaneta americana barcode sequences (n = 247, including 24 GenBank records) formed a monophyletic lineage separate from other Periplaneta species. We found three distinct P. americana haplogroups with relatively small differences within (<=0.6%) and larger differences among groups (2.4%-4.7%). This could be interpreted as indicative of multiple cryptic species. However, nuclear DNA sequences (n = 77 specimens) revealed extensive gene flow among mitochondrial haplogroups, confirming a single species. This unusual genetic pattern likely reflects multiple introductions from genetically divergent source populations, followed by interbreeding in the invasive range. Our findings highlight the need for comprehensive reference databases in DNA barcoding studies, especially when dealing with invasive populations that might be derived from multiple genetically distinct source populations.

Barcoding the butterflies of southern South America: Species delimitation efficacy, cryptic diversity and geographic patterns of divergence.

PubMed

Lavinia, Pablo D; Núñez Bustos, Ezequiel O; Kopuchian, Cecilia; Lijtmaer, Darío A; García, Natalia C; Hebert, Paul D N; Tubaro, Pablo L

2017-01-01

Because the tropical regions of America harbor the highest concentration of butterfly species, its fauna has attracted considerable attention. Much less is known about the butterflies of southern South America, particularly Argentina, where over 1,200 species occur. To advance understanding of this fauna, we assembled a DNA barcode reference library for 417 butterfly species of Argentina, focusing on the Atlantic Forest, a biodiversity hotspot. We tested the efficacy of this library for specimen identification, used it to assess the frequency of cryptic species, and examined geographic patterns of genetic variation, making this study the first large-scale genetic assessment of the butterflies of southern South America. The average sequence divergence to the nearest neighbor (i.e. minimum interspecific distance) was 6.91%, ten times larger than the mean distance to the furthest conspecific (0.69%), with a clear barcode gap present in all but four of the species represented by two or more specimens. As a consequence, the DNA barcode library was extremely effective in the discrimination of these species, allowing a correct identification in more than 95% of the cases. Singletons (i.e. species represented by a single sequence) were also distinguishable in the gene trees since they all had unique DNA barcodes, divergent from those of the closest non-conspecific. The clustering algorithms implemented recognized from 416 to 444 barcode clusters, suggesting that the actual diversity of butterflies in Argentina is 3%-9% higher than currently recognized. Furthermore, our survey added three new records of butterflies for the country (Eurema agave, Mithras hannelore, Melanis hillapana). In summary, this study not only supported the utility of DNA barcoding for the identification of the butterfly species of Argentina, but also highlighted several cases of both deep intraspecific and shallow interspecific divergence that should be studied in more detail.

Barcoding the butterflies of southern South America: Species delimitation efficacy, cryptic diversity and geographic patterns of divergence

PubMed Central

Núñez Bustos, Ezequiel O.; Kopuchian, Cecilia; Lijtmaer, Darío A.; García, Natalia C.; Hebert, Paul D. N.; Tubaro, Pablo L.

2017-01-01

Because the tropical regions of America harbor the highest concentration of butterfly species, its fauna has attracted considerable attention. Much less is known about the butterflies of southern South America, particularly Argentina, where over 1,200 species occur. To advance understanding of this fauna, we assembled a DNA barcode reference library for 417 butterfly species of Argentina, focusing on the Atlantic Forest, a biodiversity hotspot. We tested the efficacy of this library for specimen identification, used it to assess the frequency of cryptic species, and examined geographic patterns of genetic variation, making this study the first large-scale genetic assessment of the butterflies of southern South America. The average sequence divergence to the nearest neighbor (i.e. minimum interspecific distance) was 6.91%, ten times larger than the mean distance to the furthest conspecific (0.69%), with a clear barcode gap present in all but four of the species represented by two or more specimens. As a consequence, the DNA barcode library was extremely effective in the discrimination of these species, allowing a correct identification in more than 95% of the cases. Singletons (i.e. species represented by a single sequence) were also distinguishable in the gene trees since they all had unique DNA barcodes, divergent from those of the closest non-conspecific. The clustering algorithms implemented recognized from 416 to 444 barcode clusters, suggesting that the actual diversity of butterflies in Argentina is 3%–9% higher than currently recognized. Furthermore, our survey added three new records of butterflies for the country (Eurema agave, Mithras hannelore, Melanis hillapana). In summary, this study not only supported the utility of DNA barcoding for the identification of the butterfly species of Argentina, but also highlighted several cases of both deep intraspecific and shallow interspecific divergence that should be studied in more detail. PMID:29049373

Development of High Throughput Process for Constructing 454 Titanium and Illumina Libraries

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deshpande, Shweta; Hack, Christopher; Tang, Eric

2010-05-28

We have developed two processes with the Biomek FX robot to construct 454 titanium and Illumina libraries in order to meet the increasing library demands. All modifications in the library construction steps were made to enable the adaptation of the entire processes to work with the 96-well plate format. The key modifications include the shearing of DNA with Covaris E210 and the enzymatic reaction cleaning and fragment size selection with SPRI beads and magnetic plate holders. The construction of 96 Titanium libraries takes about 8 hours from sheared DNA to ssDNA recovery. The processing of 96 Illumina libraries takes lessmore » time than that of the Titanium library process. Although both processes still require manual transfer of plates from robot to other work stations such as thermocyclers, these robotic processes represent about 12- to 24-folds increase of library capacity comparing to the manual processes. To enable the sequencing of many libraries in parallel, we have also developed sets of molecular barcodes for both library types. The requirements for the 454 library barcodes include 10 bases, 40-60percent GC, no consecutive same base, and no less than 3 bases difference between barcodes. We have used 96 of the resulted 270 barcodes to construct libraries and pool to test the ability of accurately assigning reads to the right samples. When allowing 1 base error occurred in the 10 base barcodes, we could assign 99.6percent of the total reads and 100percent of them were uniquely assigned. As for the Illumina barcodes, the requirements include 4 bases, balanced GC, and at least 2 bases difference between barcodes. We have begun to assess the ability to assign reads after pooling different number of libraries. We will discuss the progress and the challenges of these scale-up processes.« less

Generation and analysis of a barcode-tagged insertion mutant library in the fission yeast Schizosaccharomyces pombe

PubMed Central

2012-01-01

Background Barcodes are unique DNA sequence tags that can be used to specifically label individual mutants. The barcode-tagged open reading frame (ORF) haploid deletion mutant collections in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe allow for high-throughput mutant phenotyping because the relative growth of mutants in a population can be determined by monitoring the proportions of their associated barcodes. While these mutant collections have greatly facilitated genome-wide studies, mutations in essential genes are not present, and the roles of these genes are not as easily studied. To further support genome-scale research in S. pombe, we generated a barcode-tagged fission yeast insertion mutant library that has the potential of generating viable mutations in both essential and non-essential genes and can be easily analyzed using standard molecular biological techniques. Results An insertion vector containing a selectable ura4+ marker and a random barcode was used to generate a collection of 10,000 fission yeast insertion mutants stored individually in 384-well plates and as six pools of mixed mutants. Individual barcodes are flanked by Sfi I recognition sites and can be oligomerized in a unique orientation to facilitate barcode sequencing. Independent genetic screens on a subset of mutants suggest that this library contains a diverse collection of single insertion mutations. We present several approaches to determine insertion sites. Conclusions This collection of S. pombe barcode-tagged insertion mutants is well-suited for genome-wide studies. Because insertion mutations may eliminate, reduce or alter the function of essential and non-essential genes, this library will contain strains with a wide range of phenotypes that can be assayed by their associated barcodes. The design of the barcodes in this library allows for barcode sequencing using next generation or standard benchtop cloning approaches. PMID:22554201

Identification of species based on DNA barcode using k-mer feature vector and Random forest classifier.

PubMed

Meher, Prabina Kumar; Sahu, Tanmaya Kumar; Rao, A R

2016-11-05

DNA barcoding is a molecular diagnostic method that allows automated and accurate identification of species based on a short and standardized fragment of DNA. To this end, an attempt has been made in this study to develop a computational approach for identifying the species by comparing its barcode with the barcode sequence of known species present in the reference library. Each barcode sequence was first mapped onto a numeric feature vector based on k-mer frequencies and then Random forest methodology was employed on the transformed dataset for species identification. The proposed approach outperformed similarity-based, tree-based, diagnostic-based approaches and found comparable with existing supervised learning based approaches in terms of species identification success rate, while compared using real and simulated datasets. Based on the proposed approach, an online web interface SPIDBAR has also been developed and made freely available at http://cabgrid.res.in:8080/spidbar/ for species identification by the taxonomists. Copyright © 2016 Elsevier B.V. All rights reserved.

Great Lakes DNA barcode reference library: Mollusca, annelida, and minor phyla

EPA Science Inventory

In recent years, the research and development of DNA-based tools has improved both their sensitivity and costs. This technology has the potential to be useful in the early detection of aquatic invasive species, and can increase the scope of surveillance compared with traditional ...

Raman Barcode for Counterfeit Drug Product Detection.

PubMed

Lawson, Latevi S; Rodriguez, Jason D

2016-05-03

Potential infiltration of counterfeit drug products-containing the wrong or no active pharmaceutical ingredient (API)-into the bona fide drug supply poses a significant threat to consumers worldwide. Raman spectroscopy offers a rapid, nondestructive avenue to screen a high throughput of samples. Traditional qualitative Raman identification is typically done with spectral correlation methods that compare the spectrum of a reference sample to an unknown. This is often effective for pure materials but is quite challenging when dealing with drug products that contain different formulations of active and inactive ingredients. Typically, reliable identification of drug products using common spectral correlation algorithms can only be made if the specific product under study is present in the library of reference spectra, thereby limiting the scope of products that can be screened. In this paper, we introduce the concept of the Raman barcode for identification of drug products by comparing the known peaks in the API reference spectrum to the peaks present in the finished drug product under study. This method requires the transformation of the Raman spectra of both API and finished drug products into a barcode representation by assigning zero intensity to every spectral frequency except the frequencies that correspond to Raman peaks. By comparing the percentage of nonzero overlap between the expected API barcode and finished drug product barcode, the identity of API present can be confirmed. In this study, 18 approved finished drug products and nine simulated counterfeits were successfully identified with 100% accuracy utilizing this method.

Taxonomic challenges in freshwater fishes: a mismatch between morphology and DNA barcoding in fish of the north-eastern part of the Congo basin.

PubMed

Decru, Eva; Moelants, Tuur; De Gelas, Koen; Vreven, Emmanuel; Verheyen, Erik; Snoeks, Jos

2016-01-01

This study evaluates the utility of DNA barcoding to traditional morphology-based species identifications for the fish fauna of the north-eastern Congo basin. We compared DNA sequences (COI) of 821 samples from 206 morphologically identified species. Best match, best close match and all species barcoding analyses resulted in a rather low identification success of 87.5%, 84.5% and 64.1%, respectively. The ratio 'nearest-neighbour distance/maximum intraspecific divergence' was lower than 1 for 26.1% of the samples, indicating possible taxonomic problems. In ten genera, belonging to six families, the number of species inferred from mtDNA data exceeded the number of species identified using morphological features; and in four cases indications of possible synonymy were detected. Finally, the DNA barcodes confirmed previously known identification problems within certain genera of the Clariidae, Cyprinidae and Mormyridae. Our results underscore the large number of taxonomic problems lingering in the taxonomy of the fish fauna of the Congo basin and illustrate why DNA barcodes will contribute to future efforts to compile a reliable taxonomic inventory of the Congo basin fish fauna. Therefore, the obtained barcodes were deposited in the reference barcode library of the Barcode of Life Initiative. © 2015 John Wiley & Sons Ltd.

Design of 240,000 orthogonal 25mer DNA barcode probes.

PubMed

Xu, Qikai; Schlabach, Michael R; Hannon, Gregory J; Elledge, Stephen J

2009-02-17

DNA barcodes linked to genetic features greatly facilitate screening these features in pooled formats using microarray hybridization, and new tools are needed to design large sets of barcodes to allow construction of large barcoded mammalian libraries such as shRNA libraries. Here we report a framework for designing large sets of orthogonal barcode probes. We demonstrate the utility of this framework by designing 240,000 barcode probes and testing their performance by hybridization. From the test hybridizations, we also discovered new probe design rules that significantly reduce cross-hybridization after their introduction into the framework of the algorithm. These rules should improve the performance of DNA microarray probe designs for many applications.

Design of 240,000 orthogonal 25mer DNA barcode probes

PubMed Central

Xu, Qikai; Schlabach, Michael R.; Hannon, Gregory J.; Elledge, Stephen J.

2009-01-01

DNA barcodes linked to genetic features greatly facilitate screening these features in pooled formats using microarray hybridization, and new tools are needed to design large sets of barcodes to allow construction of large barcoded mammalian libraries such as shRNA libraries. Here we report a framework for designing large sets of orthogonal barcode probes. We demonstrate the utility of this framework by designing 240,000 barcode probes and testing their performance by hybridization. From the test hybridizations, we also discovered new probe design rules that significantly reduce cross-hybridization after their introduction into the framework of the algorithm. These rules should improve the performance of DNA microarray probe designs for many applications. PMID:19171886

Linking adults and immatures of South African marine fishes.

PubMed

Steinke, Dirk; Connell, Allan D; Hebert, Paul D N

2016-11-01

The early life-history stages of fishes are poorly known, impeding acquisition of the identifications needed to monitor larval recruitment and year-class strength. A comprehensive database of COI sequences, linked to authoritatively identified voucher specimens, promises to change this situation, representing a significant advance for fisheries science. Barcode records were obtained from 2526 early larvae and pelagic eggs of fishes collected on the inshore shelf within 5 km of the KwaZulu-Natal coast, about 50 km south of Durban, South Africa. Barcodes were also obtained from 3215 adults, representing 946 South African fish species. Using the COI reference library on BOLD based on adults, 89% of the immature fishes could be identified to a species level; they represented 450 species. Most of the uncertain sequences could be assigned to a genus, family, or order; only 92 specimens (4%) were unassigned. Accumulation curves based on inference of phylogenetic diversity indicate near-completeness of the collecting effort. The entire set of adult and larval fishes included 1006 species, representing 43% of all fish species known from South African waters. However, this total included 189 species not previously recorded from this region. The fact that almost 90% of the immatures gained a species identification demonstrates the power and completeness of the DNA barcode reference library for fishes generated during the 10 years of FishBOL.

DNA barcode analysis of butterfly species from Pakistan points towards regional endemism

PubMed Central

Ashfaq, Muhammad; Akhtar, Saleem; Khan, Arif M; Adamowicz, Sarah J; Hebert, Paul D N

2013-01-01

DNA barcodes were obtained for 81 butterfly species belonging to 52 genera from sites in north-central Pakistan to test the utility of barcoding for their identification and to gain a better understanding of regional barcode variation. These species represent 25% of the butterfly fauna of Pakistan and belong to five families, although the Nymphalidae were dominant, comprising 38% of the total specimens. Barcode analysis showed that maximum conspecific divergence was 1.6%, while there was 1.7–14.3% divergence from the nearest neighbour species. Barcode records for 55 species showed <2% sequence divergence to records in the Barcode of Life Data Systems (BOLD), but only 26 of these cases involved specimens from neighbouring India and Central Asia. Analysis revealed that most species showed little incremental sequence variation when specimens from other regions were considered, but a threefold increase was noted in a few cases. There was a clear gap between maximum intraspecific and minimum nearest neighbour distance for all 81 species. Neighbour-joining cluster analysis showed that members of each species formed a monophyletic cluster with strong bootstrap support. The barcode results revealed two provisional species that could not be clearly linked to known taxa, while 24 other species gained their first coverage. Future work should extend the barcode reference library to include all butterfly species from Pakistan as well as neighbouring countries to gain a better understanding of regional variation in barcode sequences in this topographically and climatically complex region. PMID:23789612

DNA Barcode Analysis of Thrips (Thysanoptera) Diversity in Pakistan Reveals Cryptic Species Complexes.

PubMed

Iftikhar, Romana; Ashfaq, Muhammad; Rasool, Akhtar; Hebert, Paul D N

2016-01-01

Although thrips are globally important crop pests and vectors of viral disease, species identifications are difficult because of their small size and inconspicuous morphological differences. Sequence variation in the mitochondrial COI-5' (DNA barcode) region has proven effective for the identification of species in many groups of insect pests. We analyzed barcode sequence variation among 471 thrips from various plant hosts in north-central Pakistan. The Barcode Index Number (BIN) system assigned these sequences to 55 BINs, while the Automatic Barcode Gap Discovery detected 56 partitions, a count that coincided with the number of monophyletic lineages recognized by Neighbor-Joining analysis and Bayesian inference. Congeneric species showed an average of 19% sequence divergence (range = 5.6% - 27%) at COI, while intraspecific distances averaged 0.6% (range = 0.0% - 7.6%). BIN analysis suggested that all intraspecific divergence >3.0% actually involved a species complex. In fact, sequences for three major pest species (Haplothrips reuteri, Thrips palmi, Thrips tabaci), and one predatory thrips (Aeolothrips intermedius) showed deep intraspecific divergences, providing evidence that each is a cryptic species complex. The study compiles the first barcode reference library for the thrips of Pakistan, and examines global haplotype diversity in four important pest thrips.

The fish diversity in the upper reaches of the Salween River, Nujiang River, revealed by DNA barcoding.

PubMed

Chen, Weitao; Ma, Xiuhui; Shen, Yanjun; Mao, Yuntao; He, Shunping

2015-11-30

Nujiang River (NR), an essential component of the biodiversity hotspot of the Mountains of Southwest China, possesses a characteristic fish fauna and contains endemic species. Although previous studies on fish diversity in the NR have primarily consisted of listings of the fish species observed during field collections, in our study, we DNA-barcoded 1139 specimens belonging to 46 morphologically distinct fish species distributed throughout the NR basin by employing multiple analytical approaches. According to our analyses, DNA barcoding is an efficient method for the identification of fish by the presence of barcode gaps. However, three invasive species are characterized by deep conspecific divergences, generating multiple lineages and Operational Taxonomic Units (OTUs), implying the possibility of cryptic species. At the other end of the spectrum, ten species (from three genera) that are characterized by an overlap between their intra- and interspecific genetic distances form a single genetic cluster and share haplotypes. The neighbor-joining phenogram, Barcode Index Numbers (BINs) and Automatic Barcode Gap Discovery (ABGD) identified 43 putative species, while the General Mixed Yule-coalescence (GMYC) identified five more OTUs. Thus, our study established a reliable DNA barcode reference library for the fish in the NR and sheds new light on the local fish diversity.

The fish diversity in the upper reaches of the Salween River, Nujiang River, revealed by DNA barcoding

PubMed Central

Chen, Weitao; Ma, Xiuhui; Shen, Yanjun; Mao, Yuntao; He, Shunping

2015-01-01

Nujiang River (NR), an essential component of the biodiversity hotspot of the Mountains of Southwest China, possesses a characteristic fish fauna and contains endemic species. Although previous studies on fish diversity in the NR have primarily consisted of listings of the fish species observed during field collections, in our study, we DNA-barcoded 1139 specimens belonging to 46 morphologically distinct fish species distributed throughout the NR basin by employing multiple analytical approaches. According to our analyses, DNA barcoding is an efficient method for the identification of fish by the presence of barcode gaps. However, three invasive species are characterized by deep conspecific divergences, generating multiple lineages and Operational Taxonomic Units (OTUs), implying the possibility of cryptic species. At the other end of the spectrum, ten species (from three genera) that are characterized by an overlap between their intra- and interspecific genetic distances form a single genetic cluster and share haplotypes. The neighbor-joining phenogram, Barcode Index Numbers (BINs) and Automatic Barcode Gap Discovery (ABGD) identified 43 putative species, while the General Mixed Yule-coalescence (GMYC) identified five more OTUs. Thus, our study established a reliable DNA barcode reference library for the fish in the NR and sheds new light on the local fish diversity. PMID:26616046

DNA barcode data accurately assign higher spider taxa

PubMed Central

Coddington, Jonathan A.; Agnarsson, Ingi; Cheng, Ren-Chung; Čandek, Klemen; Driskell, Amy; Frick, Holger; Gregorič, Matjaž; Kostanjšek, Rok; Kropf, Christian; Kweskin, Matthew; Lokovšek, Tjaša; Pipan, Miha; Vidergar, Nina

2016-01-01

The use of unique DNA sequences as a method for taxonomic identification is no longer fundamentally controversial, even though debate continues on the best markers, methods, and technology to use. Although both existing databanks such as GenBank and BOLD, as well as reference taxonomies, are imperfect, in best case scenarios “barcodes” (whether single or multiple, organelle or nuclear, loci) clearly are an increasingly fast and inexpensive method of identification, especially as compared to manual identification of unknowns by increasingly rare expert taxonomists. Because most species on Earth are undescribed, a complete reference database at the species level is impractical in the near term. The question therefore arises whether unidentified species can, using DNA barcodes, be accurately assigned to more inclusive groups such as genera and families—taxonomic ranks of putatively monophyletic groups for which the global inventory is more complete and stable. We used a carefully chosen test library of CO1 sequences from 49 families, 313 genera, and 816 species of spiders to assess the accuracy of genus and family-level assignment. We used BLAST queries of each sequence against the entire library and got the top ten hits. The percent sequence identity was reported from these hits (PIdent, range 75–100%). Accurate assignment of higher taxa (PIdent above which errors totaled less than 5%) occurred for genera at PIdent values >95 and families at PIdent values ≥ 91, suggesting these as heuristic thresholds for accurate generic and familial identifications in spiders. Accuracy of identification increases with numbers of species/genus and genera/family in the library; above five genera per family and fifteen species per genus all higher taxon assignments were correct. We propose that using percent sequence identity between conventional barcode sequences may be a feasible and reasonably accurate method to identify animals to family/genus. However, the quality of the underlying database impacts accuracy of results; many outliers in our dataset could be attributed to taxonomic and/or sequencing errors in BOLD and GenBank. It seems that an accurate and complete reference library of families and genera of life could provide accurate higher level taxonomic identifications cheaply and accessibly, within years rather than decades. PMID:27547527

Linking Project Procedure Manual for Using Dumb-Barcode Linking on GEAC.

ERIC Educational Resources Information Center

Condron, Lyn

This procedure manual is designed to assist cataloging staff members at a university library through the 10-step process of barcoding and linking books classified by the Library of Congress system to the library's GEAC online computer system. A brief introduction provides background information on the project. The procedures involved in each…

DNA reference libraries of French Guianese mosquitoes for barcoding and metabarcoding

PubMed Central

Leroy, Céline; Guidez, Amandine; Dusfour, Isabelle; Girod, Romain; Dejean, Alain; Murienne, Jérôme

2017-01-01

The mosquito family (Diptera: Culicidae) constitutes the most medically important group of arthropods because certain species are vectors of human pathogens. In some parts of the world, the diversity is so high that the accurate delimitation and/or identification of species is challenging. A DNA-based identification system for all animals has been proposed, the so-called DNA barcoding approach. In this study, our objectives were (i) to establish DNA barcode libraries for the mosquitoes of French Guiana based on the COI and the 16S markers, (ii) to compare distance-based and tree-based methods of species delimitation to traditional taxonomy, and (iii) to evaluate the accuracy of each marker in identifying specimens. A total of 266 specimens belonging to 75 morphologically identified species or morphospecies were analyzed allowing us to delimit 86 DNA clusters with only 21 of them already present in the BOLD database. We thus provide a substantial contribution to the global mosquito barcoding initiative. Our results confirm that DNA barcodes can be successfully used to delimit and identify mosquito species with only a few cases where the marker could not distinguish closely related species. Our results also validate the presence of new species identified based on morphology, plus potential cases of cryptic species. We found that both COI and 16S markers performed very well, with successful identifications at the species level of up to 98% for COI and 97% for 16S when compared to traditional taxonomy. This shows great potential for the use of metabarcoding for vector monitoring and eco-epidemiological studies. PMID:28575090

Revisiting the ichthyodiversity of Java and Bali through DNA barcodes: taxonomic coverage, identification accuracy, cryptic diversity and identification of exotic species.

PubMed

Dahruddin, Hadi; Hutama, Aditya; Busson, Frédéric; Sauri, Sopian; Hanner, Robert; Keith, Philippe; Hadiaty, Renny; Hubert, Nicolas

2017-03-01

Among the 899 species of freshwater fishes reported from Sundaland biodiversity hotspot, nearly 50% are endemics. The functional integrity of aquatic ecosystems is currently jeopardized by human activities, and landscape conversion led to the decline of fish populations in several part of Sundaland, particularly in Java. The inventory of the Javanese ichthyofauna has been discontinuous, and the taxonomic knowledge is scattered in the literature. This study provides a DNA barcode reference library for the inland fishes of Java and Bali with the aim to streamline the inventory of fishes in this part of Sundaland. Owing to the lack of available checklist for estimating the taxonomic coverage of this study, a checklist was compiled based on online catalogues. A total of 95 sites were visited, and a library including 1046 DNA barcodes for 159 species was assembled. Nearest neighbour distance was 28-fold higher than maximum intraspecific distance on average, and a DNA barcoding gap was observed. The list of species with DNA barcodes displayed large discrepancies with the checklist compiled here as only 36% (i.e. 77 species) and 60% (i.e. 24 species) of the known species were sampled in Java and Bali, respectively. This result was contrasted by a high number of new occurrences and the ceiling of the accumulation curves for both species and genera. These results highlight the poor taxonomic knowledge of this ichthyofauna, and the apparent discrepancy between present and historical occurrence data is to be attributed to species extirpations, synonymy and misidentifications in previous studies. © 2016 John Wiley & Sons Ltd.

Towards a global barcode library for Lymantria (Lepidoptera: Lymantriinae) tussock moths of biosecurity concern.

PubMed

deWaard, Jeremy R; Mitchell, Andrew; Keena, Melody A; Gopurenko, David; Boykin, Laura M; Armstrong, Karen F; Pogue, Michael G; Lima, Joao; Floyd, Robin; Hanner, Robert H; Humble, Leland M

2010-12-09

Detecting and controlling the movements of invasive species, such as insect pests, relies upon rapid and accurate species identification in order to initiate containment procedures by the appropriate authorities. Many species in the tussock moth genus Lymantria are significant forestry pests, including the gypsy moth Lymantria dispar L., and consequently have been a focus for the development of molecular diagnostic tools to assist in identifying species and source populations. In this study we expand the taxonomic and geographic coverage of the DNA barcode reference library, and further test the utility of this diagnostic method, both for species/subspecies assignment and for determination of geographic provenance of populations. Cytochrome oxidase I (COI) barcodes were obtained from 518 individuals and 36 species of Lymantria, including sequences assembled and generated from previous studies, vouchered material in public collections, and intercepted specimens obtained from surveillance programs in Canada. A maximum likelihood tree was constructed, revealing high bootstrap support for 90% of species clusters. Bayesian species assignment was also tested, and resulted in correct assignment to species and subspecies in all instances. The performance of barcoding was also compared against the commonly employed NB restriction digest system (also based on COI); while the latter is informative for discriminating gypsy moth subspecies, COI barcode sequences provide greater resolution and generality by encompassing a greater number of haplotypes across all Lymantria species, none shared between species. This study demonstrates the efficacy of DNA barcodes for diagnosing species of Lymantria and reinforces the view that the approach is an under-utilized resource with substantial potential for biosecurity and surveillance. Biomonitoring agencies currently employing the NB restriction digest system would gather more information by transitioning to the use of DNA barcoding, a change which could be made relatively seamlessly as the same gene region underlies both protocols.

DNA barcode analysis of butterfly species from Pakistan points towards regional endemism.

PubMed

Ashfaq, Muhammad; Akhtar, Saleem; Khan, Arif M; Adamowicz, Sarah J; Hebert, Paul D N

2013-09-01

DNA barcodes were obtained for 81 butterfly species belonging to 52 genera from sites in north-central Pakistan to test the utility of barcoding for their identification and to gain a better understanding of regional barcode variation. These species represent 25% of the butterfly fauna of Pakistan and belong to five families, although the Nymphalidae were dominant, comprising 38% of the total specimens. Barcode analysis showed that maximum conspecific divergence was 1.6%, while there was 1.7-14.3% divergence from the nearest neighbour species. Barcode records for 55 species showed <2% sequence divergence to records in the Barcode of Life Data Systems (BOLD), but only 26 of these cases involved specimens from neighbouring India and Central Asia. Analysis revealed that most species showed little incremental sequence variation when specimens from other regions were considered, but a threefold increase was noted in a few cases. There was a clear gap between maximum intraspecific and minimum nearest neighbour distance for all 81 species. Neighbour-joining cluster analysis showed that members of each species formed a monophyletic cluster with strong bootstrap support. The barcode results revealed two provisional species that could not be clearly linked to known taxa, while 24 other species gained their first coverage. Future work should extend the barcode reference library to include all butterfly species from Pakistan as well as neighbouring countries to gain a better understanding of regional variation in barcode sequences in this topographically and climatically complex region. © 2013 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.

DNA Barcode Reference Library for the African Citrus Triozid, Trioza erytreae (Hemiptera: Triozidae): Vector of African Citrus Greening.

PubMed

Khamis, F M; Rwomushana, I; Ombura, L O; Cook, G; Mohamed, S A; Tanga, C M; Nderitu, P W; Borgemeister, C; Sétamou, M; Grout, T G; Ekesi, S

2017-12-05

Citrus (Citrus spp.) production continues to decline in East Africa, particularly in Kenya and Tanzania, the two major producers in the region. This decline is attributed to pests and diseases including infestation by the African citrus triozid, Trioza erytreae (Del Guercio) (Hemiptera: Triozidae). Besides direct feeding damage by adults and immature stages, T. erytreae is the main vector of 'Candidatus Liberibacter africanus', the causative agent of Greening disease in Africa, closely related to Huanglongbing. This study aimed to generate a novel barcode reference library for T. erytreae in order to use DNA barcoding as a rapid tool for accurate identification of the pest to aid phytosanitary measures. Triozid samples were collected from citrus orchards in Kenya, Tanzania, and South Africa and from alternative host plants. Sequences generated from populations in the study showed very low variability within acceptable ranges of species. All samples analyzed were linked to T. erytreae of GenBank accession number KU517195. Phylogeny of samples in this study and other Trioza reference species was inferred using the Maximum Likelihood method. The phylogenetic tree was paraphyletic with two distinct branches. The first branch had two clusters: 1) cluster of all populations analyzed with GenBank accession of T. erytreae and 2) cluster of all the other GenBank accession of Trioza species analyzed except T. incrustata Percy, 2016 (KT588307.1), T. eugeniae Froggatt (KY294637.1), and T. grallata Percy, 2016 (KT588308.1) that occupied the second branch as outgroups forming sister clade relationships. These results were further substantiated with genetic distance values and principal component analyses. © The Author(s) 2017. Published by Oxford University Press on behalf of Entomological Society of America.

Droplet Digital™ PCR Next-Generation Sequencing Library QC Assay.

PubMed

Heredia, Nicholas J

2018-01-01

Digital PCR is a valuable tool to quantify next-generation sequencing (NGS) libraries precisely and accurately. Accurately quantifying NGS libraries enable accurate loading of the libraries on to the sequencer and thus improve sequencing performance by reducing under and overloading error. Accurate quantification also benefits users by enabling uniform loading of indexed/barcoded libraries which in turn greatly improves sequencing uniformity of the indexed/barcoded samples. The advantages gained by employing the Droplet Digital PCR (ddPCR™) library QC assay includes the precise and accurate quantification in addition to size quality assessment, enabling users to QC their sequencing libraries with confidence.

Managing Archival Collections in an Automated Environment: The Joys of Barcoding

ERIC Educational Resources Information Center

Hamburger, Susan; Charles, Jane Veronica

2006-01-01

In a desire for automated collection control, archival repositories are adopting barcoding from their library and records center colleagues. This article discusses the planning, design, and implementation phases of barcoding. The authors focus on reasons for barcoding, security benefits, in-room circulation tracking, potential for gathering…

Establishing a community-wide DNA barcode library as a new tool for arctic research.

PubMed

Wirta, H; Várkonyi, G; Rasmussen, C; Kaartinen, R; Schmidt, N M; Hebert, P D N; Barták, M; Blagoev, G; Disney, H; Ertl, S; Gjelstrup, P; Gwiazdowicz, D J; Huldén, L; Ilmonen, J; Jakovlev, J; Jaschhof, M; Kahanpää, J; Kankaanpää, T; Krogh, P H; Labbee, R; Lettner, C; Michelsen, V; Nielsen, S A; Nielsen, T R; Paasivirta, L; Pedersen, S; Pohjoismäki, J; Salmela, J; Vilkamaa, P; Väre, H; von Tschirnhaus, M; Roslin, T

2016-05-01

DNA sequences offer powerful tools for describing the members and interactions of natural communities. In this study, we establish the to-date most comprehensive library of DNA barcodes for a terrestrial site, including all known macroscopic animals and vascular plants of an intensively studied area of the High Arctic, the Zackenberg Valley in Northeast Greenland. To demonstrate its utility, we apply the library to identify nearly 20 000 arthropod individuals from two Malaise traps, each operated for two summers. Drawing on this material, we estimate the coverage of previous morphology-based species inventories, derive a snapshot of faunal turnover in space and time and describe the abundance and phenology of species in the rapidly changing arctic environment. Overall, 403 terrestrial animal and 160 vascular plant species were recorded by morphology-based techniques. DNA barcodes (CO1) offered high resolution in discriminating among the local animal taxa, with 92% of morphologically distinguishable taxa assigned to unique Barcode Index Numbers (BINs) and 93% to monophyletic clusters. For vascular plants, resolution was lower, with 54% of species forming monophyletic clusters based on barcode regions rbcLa and ITS2. Malaise catches revealed 122 BINs not detected by previous sampling and DNA barcoding. The insect community was dominated by a few highly abundant taxa. Even closely related taxa differed in phenology, emphasizing the need for species-level resolution when describing ongoing shifts in arctic communities and ecosystems. The DNA barcode library now established for Zackenberg offers new scope for such explorations, and for the detailed dissection of interspecific interactions throughout the community. © 2015 John Wiley & Sons Ltd.

Incorporating DNA barcodes into a multi-year inventory of the fishes of the hyperdiverse Lower Congo River, with a multi-gene performance assessment of the genus Labeo as a case study.

PubMed

Lowenstein, Jacob H; Osmundson, Todd W; Becker, Sven; Hanner, Robert; Stiassny, Melanie L J

2011-10-01

Here we describe preliminary efforts to integrate DNA barcoding into an ongoing inventory of the Lower Congo River (LCR) ichthyofauna. The 350 km stretch of the LCR from Pool Malebo to Boma includes the world's largest river rapids. The LCR ichthyofauna is hyperdiverse and rich in endemism due to high habitat heterogeneity, numerous dispersal barriers, and its downstream location in the basin. We have documented 328 species from the LCR, 25% of which are thought to be endemic. In addition to detailing progress made to generate a reference sequence library of DNA barcodes for these fishes, we ask how DNA can be used at the current stage of the Fish Barcode of Life initiative, as a work in progress currently of limited utility to a wide audience. Two possibilities that we explore are the potential for DNA barcodes to generate discrete diagnostic characters for species, and to help resolve problematic taxa lacking clear morphologically diagnostic characters such as many species of the cyprinid genus Labeo, which we use as a case study. Our molecular analysis helped to clarify the validity of some species that were the subject of historical debate, and we were able to construct a molecular key for all monophyletic and morphologically recognizable species. Several species sampled from across the Congo Basin and widely distributed throughout Central and West Africa were recovered as paraphyletic based on our molecular data. Our study underscores the importance of generating reference barcodes for specimens collected from, or in close proximity to, type localities, particularly where species are poorly understood taxonomically and the extent of their geographical distributions have yet to be established.

DNA Barcoding of the Endangered Aquilaria (Thymelaeaceae) and Its Application in Species Authentication of Agarwood Products Traded in the Market

PubMed Central

Lee, Shiou Yih; Ng, Wei Lun; Mahat, Mohd Noor; Nazre, Mohd; Mohamed, Rozi

2016-01-01

The identification of Aquilaria species from their resinous non-wood product, the agarwood, is challenging as conventional techniques alone are unable to ascertain the species origin. Aquilaria is a highly protected species due to the excessive exploitation of its precious agarwood. Here, we applied the DNA barcoding technique to generate barcode sequences for Aquilaria species and later applied the barcodes to identify the source species of agarwood found in the market. We developed a reference DNA barcode library using eight candidate barcode loci (matK, rbcL, rpoB, rpoC1, psbA-trnH, trnL-trnF, ITS, and ITS2) amplified from 24 leaf accessions of seven Aquilaria species obtained from living trees. Our results indicated that all single barcodes can be easily amplified and sequenced with the selected primers. The combination of trnL-trnF+ITS and trnL-trnF+ITS2 yielded the greatest species resolution using the least number of loci combination, while matK+trnL-trnF+ITS showed potential in detecting the geographical origins of Aquilaria species. We propose trnL-trnF+ITS2 as the best candidate barcode for Aquilaria as ITS2 has a shorter sequence length compared to ITS, which eases PCR amplification especially when using degraded DNA samples such as those extracted from processed agarwood products. A blind test conducted on eight agarwood samples in different forms using the proposed barcode combination proved successful in their identification up to the species level. Such potential of DNA barcoding in identifying the source species of agarwood will contribute to the international timber trade control, by providing an effective method for species identification and product authentication. PMID:27128309

The seven deadly sins of DNA barcoding.

PubMed

Collins, R A; Cruickshank, R H

2013-11-01

Despite the broad benefits that DNA barcoding can bring to a diverse range of biological disciplines, a number of shortcomings still exist in terms of the experimental design of studies incorporating this approach. One underlying reason for this lies in the confusion that often exists between species discovery and specimen identification, and this is reflected in the way that hypotheses are generated and tested. Although these aims can be associated, they are quite distinct and require different methodological approaches, but their conflation has led to the frequently inappropriate use of commonly used analytical methods such as neighbour-joining trees, bootstrap resampling and fixed distance thresholds. Furthermore, the misidentification of voucher specimens can also have serious implications for end users of reference libraries such as the Barcode of Life Data Systems, and in this regard we advocate increased diligence in the a priori identification of specimens to be used for this purpose. This commentary provides an assessment of seven deficiencies that we identify as common in the DNA barcoding literature, and outline some potential improvements for its adaptation and adoption towards more reliable and accurate outcomes. © 2012 John Wiley & Sons Ltd.

A novel process of viral vector barcoding and library preparation enables high-diversity library generation and recombination-free paired-end sequencing

PubMed Central

Davidsson, Marcus; Diaz-Fernandez, Paula; Schwich, Oliver D.; Torroba, Marcos; Wang, Gang; Björklund, Tomas

2016-01-01

Detailed characterization and mapping of oligonucleotide function in vivo is generally a very time consuming effort that only allows for hypothesis driven subsampling of the full sequence to be analysed. Recent advances in deep sequencing together with highly efficient parallel oligonucleotide synthesis and cloning techniques have, however, opened up for entirely new ways to map genetic function in vivo. Here we present a novel, optimized protocol for the generation of universally applicable, barcode labelled, plasmid libraries. The libraries are designed to enable the production of viral vector preparations assessing coding or non-coding RNA function in vivo. When generating high diversity libraries, it is a challenge to achieve efficient cloning, unambiguous barcoding and detailed characterization using low-cost sequencing technologies. With the presented protocol, diversity of above 3 million uniquely barcoded adeno-associated viral (AAV) plasmids can be achieved in a single reaction through a process achievable in any molecular biology laboratory. This approach opens up for a multitude of in vivo assessments from the evaluation of enhancer and promoter regions to the optimization of genome editing. The generated plasmid libraries are also useful for validation of sequencing clustering algorithms and we here validate the newly presented message passing clustering process named Starcode. PMID:27874090

A checklist of the bats of Peninsular Malaysia and progress towards a DNA barcode reference library.

PubMed

Lim, Voon-Ching; Ramli, Rosli; Bhassu, Subha; Wilson, John-James

2017-01-01

Several published checklists of bat species have covered Peninsular Malaysia as part of a broader region and/or in combination with other mammal groups. Other researchers have produced comprehensive checklists for specific localities within the peninsula. To our knowledge, a comprehensive checklist of bats specifically for the entire geopolitical region of Peninsular Malaysia has never been published, yet knowing which species are present in Peninsular Malaysia and their distributions across the region are crucial in developing suitable conservation plans. Our literature search revealed that 110 bat species have been documented in Peninsular Malaysia; 105 species have precise locality records while five species lack recent and/or precise locality records. We retrieved 18 species from records dated before the year 2000 and seven species have only ever been recorded once. Our search of Barcode of Life Datasystems (BOLD) found that 86 (of the 110) species have public records of which 48 species have public DNA barcodes available from bats sampled in Peninsular Malaysia. Based on Neighbour-Joining tree analyses and the allocation of DNA barcodes to Barcode Index Number system (BINs) by BOLD, several DNA barcodes recorded under the same species name are likely to represent distinct taxa. We discuss these cases in detail and highlight the importance of further surveys to determine the occurences and resolve the taxonomy of particular bat species in Peninsular Malaysia, with implications for conservation priorities.

DNA barcoding detects contamination and substitution in North American herbal products

PubMed Central

2013-01-01

Background Herbal products available to consumers in the marketplace may be contaminated or substituted with alternative plant species and fillers that are not listed on the labels. According to the World Health Organization, the adulteration of herbal products is a threat to consumer safety. Our research aimed to investigate herbal product integrity and authenticity with the goal of protecting consumers from health risks associated with product substitution and contamination. Methods We used DNA barcoding to conduct a blind test of the authenticity for (i) 44 herbal products representing 12 companies and 30 different species of herbs, and (ii) 50 leaf samples collected from 42 herbal species. Our laboratory also assembled the first standard reference material (SRM) herbal barcode library from 100 herbal species of known provenance that were used to identify the unknown herbal products and leaf samples. Results We recovered DNA barcodes from most herbal products (91%) and all leaf samples (100%), with 95% species resolution using a tiered approach (rbcL + ITS2). Most (59%) of the products tested contained DNA barcodes from plant species not listed on the labels. Although we were able to authenticate almost half (48%) of the products, one-third of these also contained contaminants and or fillers not listed on the label. Product substitution occurred in 30/44 of the products tested and only 2/12 companies had products without any substitution, contamination or fillers. Some of the contaminants we found pose serious health risks to consumers. Conclusions Most of the herbal products tested were of poor quality, including considerable product substitution, contamination and use of fillers. These activities dilute the effectiveness of otherwise useful remedies, lowering the perceived value of all related products because of a lack of consumer confidence in them. We suggest that the herbal industry should embrace DNA barcoding for authenticating herbal products through testing of raw materials used in manufacturing products. The use of an SRM DNA herbal barcode library for testing bulk materials could provide a method for 'best practices? in the manufacturing of herbal products. This would provide consumers with safe, high quality herbal products. PMID:24120035

From water striders to water bugs: the molecular diversity of aquatic Heteroptera (Gerromorpha, Nepomorpha) of Germany based on DNA barcodes

PubMed Central

Havemann, Nadine; Gossner, Martin M.; Hendrich, Lars; Morinière, Jèrôme; Niedringhaus, Rolf; Schäfer, Peter

2018-01-01

With about 5,000 species worldwide, the Heteroptera or true bugs are the most diverse taxon among the hemimetabolous insects in aquatic and semi-aquatic ecosystems. Species may be found in almost every freshwater environment and have very specific habitat requirements, making them excellent bioindicator organisms for water quality. However, a correct determination by morphology is challenging in many species groups due to high morphological variability and polymorphisms within, but low variability between species. Furthermore, it is very difficult or even impossible to identify the immature life stages or females of some species, e.g., of the corixid genus Sigara. In this study we tested the effectiveness of a DNA barcode library to discriminate species of the Gerromorpha and Nepomorpha of Germany. We analyzed about 700 specimens of 67 species, with 63 species sampled in Germany, covering more than 90% of all recorded species. Our library included various morphological similar taxa, e.g., species within the genera Sigara and Notonecta as well as water striders of the genus Gerris. Fifty-five species (82%) were unambiguously assigned to a single Barcode Index Number (BIN) by their barcode sequences, whereas BIN sharing was observed for 10 species. Furthermore, we found monophyletic lineages for 52 analyzed species. Our data revealed interspecific K2P distances with below 2.2% for 18 species. Intraspecific distances above 2.2% were shown for 11 species. We found evidence for hybridization between various corixid species (Sigara, Callicorixa), but our molecular data also revealed exceptionally high intraspecific distances as a consequence of distinct mitochondrial lineages for Cymatia coleoptrata and the pygmy backswimmer Plea minutissima. Our study clearly demonstrates the usefulness of DNA barcodes for the identification of the aquatic Heteroptera of Germany and adjacent regions. In this context, our data set represents an essential baseline for a reference library for bioassessment studies of freshwater habitats using modern high-throughput technologies in the near future. The existing data also opens new questions regarding the causes of observed low inter- and high intraspecific genetic variation and furthermore highlight the necessity of taxonomic revisions for various taxa, combining both molecular and morphological data. PMID:29736329

DNA Barcoding of Freshwater Fishes of Indo-Myanmar Biodiversity Hotspot.

PubMed

Barman, Anindya Sundar; Singh, Mamta; Singh, Soibam Khogen; Saha, Himadri; Singh, Yumlembam Jackie; Laishram, Martina; Pandey, Pramod Kumar

2018-06-05

To develop an effective conservation and management strategy, it is required to assess the biodiversity status of an ecosystem, especially when we deal with Indo-Myanmar biodiversity hotspot. Importance of this reaches to an entirely different level as the hotspot represents the area of high endemism which is under continuous threat. Therefore, the need of the present study was conceptualized, dealing with molecular assessment of the fish fauna of Indo-Myanmar region, which covers the Indian states namely, Manipur, Meghalaya, Mizoram, and Nagaland. A total of 363 specimens, representing 109 species were collected and barcoded from the different rivers and their tributaries of the region. The analyses performed in the present study, i.e. Kimura 2-Parameter genetic divergence, Neighbor-Joining, Automated Barcode Gap Discovery and Bayesian Poisson Tree Processes suggest that DNA barcoding is an efficient and reliable tool for species identification. Most of the species were clearly delineated. However, presence of intra-specific and inter-specific genetic distance overlap in few species, revealed the existence of putative cryptic species. A reliable DNA barcode reference library, established in our study provides an adequate knowledge base to the groups of non-taxonomists, researchers, biodiversity managers and policy makers in sketching effective conservation measures for this ecosystem.

Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing.

PubMed

Ståhlberg, Anders; Krzyzanowski, Paul M; Jackson, Jennifer B; Egyud, Matthew; Stein, Lincoln; Godfrey, Tony E

2016-06-20

Detection of cell-free DNA in liquid biopsies offers great potential for use in non-invasive prenatal testing and as a cancer biomarker. Fetal and tumor DNA fractions however can be extremely low in these samples and ultra-sensitive methods are required for their detection. Here, we report an extremely simple and fast method for introduction of barcodes into DNA libraries made from 5 ng of DNA. Barcoded adapter primers are designed with an oligonucleotide hairpin structure to protect the molecular barcodes during the first rounds of polymerase chain reaction (PCR) and prevent them from participating in mis-priming events. Our approach enables high-level multiplexing and next-generation sequencing library construction with flexible library content. We show that uniform libraries of 1-, 5-, 13- and 31-plex can be generated. Utilizing the barcodes to generate consensus reads for each original DNA molecule reduces background sequencing noise and allows detection of variant alleles below 0.1% frequency in clonal cell line DNA and in cell-free plasma DNA. Thus, our approach bridges the gap between the highly sensitive but specific capabilities of digital PCR, which only allows a limited number of variants to be analyzed, with the broad target capability of next-generation sequencing which traditionally lacks the sensitivity to detect rare variants. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Use of a rep-PCR system to predict species in the Aspergillus section Nigri.

PubMed

Palencia, Edwin R; Klich, Maren A; Glenn, Anthony E; Bacon, Charles W

2009-10-01

The Aspergillus niger aggregate within the A. section Nigri is a group of black-spored aspergilli of great agro-economic importance whose well defined taxonomy has been elusive. Rep-PCR has become a rapid and cost-effective method for genotyping fungi and bacteria. In the present study, we evaluated the discriminatory power of a semi-automated rep-PCR barcoding system to distinguish morphotypic species and compare the results with the data obtained from ITS and partial calmodulin regions. For this purpose, 20 morphotyped black-spored Aspergillus species were used to create the A. section Nigri library in this barcoding system that served to identify 34 field isolates. A pair-wise similarity matrix was calculated using the cone-based Pearson correlation method and the dendrogram was generated by the unweighted pair group method with arithmetic mean (UPGMA), illustrating four different clustered groups: the uniseriate cluster (I), the Aspergillus carbonarius cluster (II), and. the two A. niger aggregate clusters (named III.A and III.B). Rep-PCR showed higher resolution than the ITS and the partial calmodulin gene analytical procedures. The data of the 34 unknown field isolates, collected from different locations in the United States, indicated that only 12% of the field isolates were >95% similar to one of the genotypes included in the A. section Nigri library. However, 64% of the field isolates matched genotypes with the reference library (similarity values >90%). Based on these results, this barcoding procedure has the potential for use as a reproducible tool for identifying the black-spored aspergilli.

Direct Identification of On-Bead Peptides Using Surface-Enhanced Raman Spectroscopic Barcoding System for High-Throughput Bioanalysis

PubMed Central

Kang, Homan; Jeong, Sinyoung; Koh, Yul; Geun Cha, Myeong; Yang, Jin-Kyoung; Kyeong, San; Kim, Jaehi; Kwak, Seon-Yeong; Chang, Hye-Jin; Lee, Hyunmi; Jeong, Cheolhwan; Kim, Jong-Ho; Jun, Bong-Hyun; Kim, Yong-Kweon; Hong Jeong, Dae; Lee, Yoon-Sik

2015-01-01

Recently, preparation and screening of compound libraries remain one of the most challenging tasks in drug discovery, biomarker detection, and biomolecular profiling processes. So far, several distinct encoding/decoding methods such as chemical encoding, graphical encoding, and optical encoding have been reported to identify those libraries. In this paper, a simple and efficient surface-enhanced Raman spectroscopic (SERS) barcoding method using highly sensitive SERS nanoparticles (SERS ID) is presented. The 44 kinds of SERS IDs were able to generate simple codes and could possibly generate more than one million kinds of codes by incorporating combinations of different SERS IDs. The barcoding method exhibited high stability and reliability under bioassay conditions. The SERS ID encoding based screening platform can identify the peptide ligand on the bead and also quantify its binding affinity for specific protein. We believe that our SERS barcoding technology is a promising method in the screening of one-bead-one-compound (OBOC) libraries for drug discovery. PMID:26017924

Direct identification of on-bead peptides using surface-enhanced Raman spectroscopic barcoding system for high-throughput bioanalysis.

PubMed

Kang, Homan; Jeong, Sinyoung; Koh, Yul; Geun Cha, Myeong; Yang, Jin-Kyoung; Kyeong, San; Kim, Jaehi; Kwak, Seon-Yeong; Chang, Hye-Jin; Lee, Hyunmi; Jeong, Cheolhwan; Kim, Jong-Ho; Jun, Bong-Hyun; Kim, Yong-Kweon; Hong Jeong, Dae; Lee, Yoon-Sik

2015-05-28

Recently, preparation and screening of compound libraries remain one of the most challenging tasks in drug discovery, biomarker detection, and biomolecular profiling processes. So far, several distinct encoding/decoding methods such as chemical encoding, graphical encoding, and optical encoding have been reported to identify those libraries. In this paper, a simple and efficient surface-enhanced Raman spectroscopic (SERS) barcoding method using highly sensitive SERS nanoparticles (SERS ID) is presented. The 44 kinds of SERS IDs were able to generate simple codes and could possibly generate more than one million kinds of codes by incorporating combinations of different SERS IDs. The barcoding method exhibited high stability and reliability under bioassay conditions. The SERS ID encoding based screening platform can identify the peptide ligand on the bead and also quantify its binding affinity for specific protein. We believe that our SERS barcoding technology is a promising method in the screening of one-bead-one-compound (OBOC) libraries for drug discovery.

Wolbachia and DNA barcoding insects: patterns, potential, and problems.

PubMed

Smith, M Alex; Bertrand, Claudia; Crosby, Kate; Eveleigh, Eldon S; Fernandez-Triana, Jose; Fisher, Brian L; Gibbs, Jason; Hajibabaei, Mehrdad; Hallwachs, Winnie; Hind, Katharine; Hrcek, Jan; Huang, Da-Wei; Janda, Milan; Janzen, Daniel H; Li, Yanwei; Miller, Scott E; Packer, Laurence; Quicke, Donald; Ratnasingham, Sujeevan; Rodriguez, Josephine; Rougerie, Rodolphe; Shaw, Mark R; Sheffield, Cory; Stahlhut, Julie K; Steinke, Dirk; Whitfield, James; Wood, Monty; Zhou, Xin

2012-01-01

Wolbachia is a genus of bacterial endosymbionts that impacts the breeding systems of their hosts. Wolbachia can confuse the patterns of mitochondrial variation, including DNA barcodes, because it influences the pathways through which mitochondria are inherited. We examined the extent to which these endosymbionts are detected in routine DNA barcoding, assessed their impact upon the insect sequence divergence and identification accuracy, and considered the variation present in Wolbachia COI. Using both standard PCR assays (Wolbachia surface coding protein--wsp), and bacterial COI fragments we found evidence of Wolbachia in insect total genomic extracts created for DNA barcoding library construction. When >2 million insect COI trace files were examined on the Barcode of Life Datasystem (BOLD) Wolbachia COI was present in 0.16% of the cases. It is possible to generate Wolbachia COI using standard insect primers; however, that amplicon was never confused with the COI of the host. Wolbachia alleles recovered were predominantly Supergroup A and were broadly distributed geographically and phylogenetically. We conclude that the presence of the Wolbachia DNA in total genomic extracts made from insects is unlikely to compromise the accuracy of the DNA barcode library; in fact, the ability to query this DNA library (the database and the extracts) for endosymbionts is one of the ancillary benefits of such a large scale endeavor--which we provide several examples. It is our conclusion that regular assays for Wolbachia presence and type can, and should, be adopted by large scale insect barcoding initiatives. While COI is one of the five multi-locus sequence typing (MLST) genes used for categorizing Wolbachia, there is limited overlap with the eukaryotic DNA barcode region.

Environmental Barcoding: A Next-Generation Sequencing Approach for Biomonitoring Applications Using River Benthos

PubMed Central

Hajibabaei, Mehrdad; Shokralla, Shadi; Zhou, Xin; Singer, Gregory A. C.; Baird, Donald J.

2011-01-01

Timely and accurate biodiversity analysis poses an ongoing challenge for the success of biomonitoring programs. Morphology-based identification of bioindicator taxa is time consuming, and rarely supports species-level resolution especially for immature life stages. Much work has been done in the past decade to develop alternative approaches for biodiversity analysis using DNA sequence-based approaches such as molecular phylogenetics and DNA barcoding. On-going assembly of DNA barcode reference libraries will provide the basis for a DNA-based identification system. The use of recently introduced next-generation sequencing (NGS) approaches in biodiversity science has the potential to further extend the application of DNA information for routine biomonitoring applications to an unprecedented scale. Here we demonstrate the feasibility of using 454 massively parallel pyrosequencing for species-level analysis of freshwater benthic macroinvertebrate taxa commonly used for biomonitoring. We designed our experiments in order to directly compare morphology-based, Sanger sequencing DNA barcoding, and next-generation environmental barcoding approaches. Our results show the ability of 454 pyrosequencing of mini-barcodes to accurately identify all species with more than 1% abundance in the pooled mixture. Although the approach failed to identify 6 rare species in the mixture, the presence of sequences from 9 species that were not represented by individuals in the mixture provides evidence that DNA based analysis may yet provide a valuable approach in finding rare species in bulk environmental samples. We further demonstrate the application of the environmental barcoding approach by comparing benthic macroinvertebrates from an urban region to those obtained from a conservation area. Although considerable effort will be required to robustly optimize NGS tools to identify species from bulk environmental samples, our results indicate the potential of an environmental barcoding approach for biomonitoring programs. PMID:21533287

Evaluation of single and multilocus DNA barcodes towards species delineation in complex tree genus Terminalia

PubMed Central

Mishra, Priyanka; Kumar, Amit; Nagireddy, Akshitha; Shukla, Ashutosh K.

2017-01-01

DNA barcoding is used as a universal tool for delimiting species boundaries in taxonomically challenging groups, with different plastid and nuclear regions (rbcL, matK, ITS and psbA-trnH) being recommended as primary DNA barcodes for plants. We evaluated the feasibility of using these regions in the species-rich genus Terminalia, which exhibits various overlapping morphotypes with pantropical distribution, owing to its complex taxonomy. Terminalia bellerica and T. chebula are ingredients of the famous Ayurvedic Rasayana formulation Triphala, used for detoxification and rejuvenation. High demand for extracted phytochemicals as well as the high trade value of several species renders mandatory the need for the correct identification of traded plant material. Three different analytical methods with single and multilocus barcoding regions were tested to develop a DNA barcode reference library from 222 individuals representing 41 Terminalia species. All the single barcodes tested had a lower discriminatory power than the multilocus regions, and the combination of matK+ITS had the highest resolution rate (94.44%). The average intra-specific variations (0.0188±0.0019) were less than the distance to the nearest neighbour (0.106±0.009) with matK and ITS. Distance-based Neighbour Joining analysis outperformed the character-based Maximum Parsimony method in the identification of traded species such as T. arjuna, T. chebula and T. tomentosa, which are prone to adulteration. rbcL was shown to be a highly conservative region with only 3.45% variability between all of the sequences. The recommended barcode combination, rbcL+matK, failed to perform in the genus Terminalia. Considering the complexity of resolution observed with single regions, the present study proposes the combination of matK+ITS as the most successful barcode in Terminalia. PMID:28829803

A checklist of the bats of Peninsular Malaysia and progress towards a DNA barcode reference library

PubMed Central

Ramli, Rosli; Bhassu, Subha

2017-01-01

Several published checklists of bat species have covered Peninsular Malaysia as part of a broader region and/or in combination with other mammal groups. Other researchers have produced comprehensive checklists for specific localities within the peninsula. To our knowledge, a comprehensive checklist of bats specifically for the entire geopolitical region of Peninsular Malaysia has never been published, yet knowing which species are present in Peninsular Malaysia and their distributions across the region are crucial in developing suitable conservation plans. Our literature search revealed that 110 bat species have been documented in Peninsular Malaysia; 105 species have precise locality records while five species lack recent and/or precise locality records. We retrieved 18 species from records dated before the year 2000 and seven species have only ever been recorded once. Our search of Barcode of Life Datasystems (BOLD) found that 86 (of the 110) species have public records of which 48 species have public DNA barcodes available from bats sampled in Peninsular Malaysia. Based on Neighbour-Joining tree analyses and the allocation of DNA barcodes to Barcode Index Number system (BINs) by BOLD, several DNA barcodes recorded under the same species name are likely to represent distinct taxa. We discuss these cases in detail and highlight the importance of further surveys to determine the occurences and resolve the taxonomy of particular bat species in Peninsular Malaysia, with implications for conservation priorities. PMID:28742835

DNA barcoding discriminates freshwater fishes from southeastern Nigeria and provides river system-level phylogeographic resolution within some species.

PubMed

Nwani, Christopher D; Becker, Sven; Braid, Heather E; Ude, Emmanuel F; Okogwu, Okechukwu I; Hanner, Robert

2011-10-01

Fishes are the main animal protein source for human beings and play a vital role in aquatic ecosystems and food webs. Fish identification can be challenging, especially in the tropics (due to high diversity), and this is particularly true for larval forms or fragmentary remains. DNA barcoding, which uses the 5' region of the mitochondrial cytochrome c oxidase subunit I (COI) as a target gene, is an efficient method for standardized species-level identification for biodiversity assessment and conservation, pending the establishment of reference sequence libraries. In this study, fishes were collected from three rivers in southeastern Nigeria, identified morphologically, and imaged digitally. DNA was extracted, PCR-amplified, and the standard barcode region was bidirectionally sequenced for 363 individuals belonging to 70 species in 38 genera. All specimen provenance data and associated sequence information were recorded in the barcode of life data systems (BOLD; www.barcodinglife.org ). Analytical tools on BOLD were used to assess the performance of barcoding to identify species. Using neighbor-joining distance comparison, the average genetic distance was 60-fold higher between species than within species, as pairwise genetic distance estimates averaged 10.29% among congeners and only 0.17% among conspecifics. Despite low levels of divergence within species, we observed river system-specific haplotype partitioning within eight species (11.4% of all species). Our preliminary results suggest that DNA barcoding is very effective for species identification of Nigerian freshwater fishes.

Identification of the vascular plants of Churchill, Manitoba, using a DNA barcode library

PubMed Central

2012-01-01

Background Because arctic plant communities are highly vulnerable to climate change, shifts in their composition require rapid, accurate identifications, often for specimens that lack diagnostic floral characters. The present study examines the role that DNA barcoding can play in aiding floristic evaluations in the arctic by testing the effectiveness of the core plant barcode regions (rbcL, matK) and a supplemental ribosomal DNA (ITS2) marker for a well-studied flora near Churchill, Manitoba. Results This investigation examined 900 specimens representing 312 of the 354 species of vascular plants known from Churchill. Sequencing success was high for rbcL: 95% for fresh specimens and 85% for herbarium samples (mean age 20 years). ITS2 worked equally well for the fresh and herbarium material (89% and 88%). However, sequencing success was lower for matK, despite two rounds of PCR amplification, which reflected less effective primer binding and sensitivity to the DNA degradation (76% of fresh, 45% of herbaria samples). A species was considered as taxonomically resolved if its members showed at least one diagnostic difference from any other taxon in the study and formed a monophyletic clade. The highest species resolution (69%) was obtained by combining information from all three genes. The joint sequence information for rbcL and matK distinguished 54% of 286 species, while rbcL and ITS2 distinguished 63% of 285 species. Discrimination of species within Salix, which constituted 8% of the flora, was particularly problematic. Despite incomplete resolution, the barcode results revealed 22 misidentified herbarium specimens, and enabled the identification of field specimens which were otherwise too immature to identify. Although seven cases of ITS2 paralogy were noted in the families Cyperaceae, Juncaceae and Juncaginaceae, this intergenic spacer played an important role in resolving congeneric plant species at Churchill. Conclusions Our results provided fast and cost-effective solution to create a comprehensive, effective DNA barcode reference library for a local flora. PMID:23190419

Identification of the vascular plants of Churchill, Manitoba, using a DNA barcode library.

PubMed

Kuzmina, Maria L; Johnson, Karen L; Barron, Hannah R; Hebert, Paul Dn

2012-11-28

Because arctic plant communities are highly vulnerable to climate change, shifts in their composition require rapid, accurate identifications, often for specimens that lack diagnostic floral characters. The present study examines the role that DNA barcoding can play in aiding floristic evaluations in the arctic by testing the effectiveness of the core plant barcode regions (rbcL, matK) and a supplemental ribosomal DNA (ITS2) marker for a well-studied flora near Churchill, Manitoba. This investigation examined 900 specimens representing 312 of the 354 species of vascular plants known from Churchill. Sequencing success was high for rbcL: 95% for fresh specimens and 85% for herbarium samples (mean age 20 years). ITS2 worked equally well for the fresh and herbarium material (89% and 88%). However, sequencing success was lower for matK, despite two rounds of PCR amplification, which reflected less effective primer binding and sensitivity to the DNA degradation (76% of fresh, 45% of herbaria samples). A species was considered as taxonomically resolved if its members showed at least one diagnostic difference from any other taxon in the study and formed a monophyletic clade. The highest species resolution (69%) was obtained by combining information from all three genes. The joint sequence information for rbcL and matK distinguished 54% of 286 species, while rbcL and ITS2 distinguished 63% of 285 species. Discrimination of species within Salix, which constituted 8% of the flora, was particularly problematic. Despite incomplete resolution, the barcode results revealed 22 misidentified herbarium specimens, and enabled the identification of field specimens which were otherwise too immature to identify. Although seven cases of ITS2 paralogy were noted in the families Cyperaceae, Juncaceae and Juncaginaceae, this intergenic spacer played an important role in resolving congeneric plant species at Churchill. Our results provided fast and cost-effective solution to create a comprehensive, effective DNA barcode reference library for a local flora.

Implications of Hybridization, NUMTs, and Overlooked Diversity for DNA Barcoding of Eurasian Ground Squirrels

PubMed Central

Ermakov, Oleg A.; Simonov, Evgeniy; Surin, Vadim L.; Titov, Sergey V.; Brandler, Oleg V.; Ivanova, Natalia V.; Borisenko, Alex V.

2015-01-01

The utility of DNA Barcoding for species identification and discovery has catalyzed a concerted effort to build the global reference library; however, many animal groups of economical or conservational importance remain poorly represented. This study aims to contribute DNA barcode records for all ground squirrel species (Xerinae, Sciuridae, Rodentia) inhabiting Eurasia and to test efficiency of this approach for species discrimination. Cytochrome c oxidase subunit 1 (COI) gene sequences were obtained for 97 individuals representing 16 ground squirrel species of which 12 were correctly identified. Taxonomic allocation of some specimens within four species was complicated by geographically restricted mtDNA introgression. Exclusion of individuals with introgressed mtDNA allowed reaching a 91.6% identification success rate. Significant COI divergence (3.5–4.4%) was observed within the most widespread ground squirrel species (Spermophilus erythrogenys, S. pygmaeus, S. suslicus, Urocitellus undulatus), suggesting the presence of cryptic species. A single putative NUMT (nuclear mitochondrial pseudogene) sequence was recovered during molecular analysis; mitochondrial COI from this sample was amplified following re-extraction of DNA. Our data show high discrimination ability of 100 bp COI fragments for Eurasian ground squirrels (84.3%) with no incorrect assessments, underscoring the potential utility of the existing reference librariy for the development of diagnostic ‘mini-barcodes’. PMID:25617768

Tagmentation on Microbeads: Restore Long-Range DNA Sequence Information Using Next Generation Sequencing with Library Prepared by Surface-Immobilized Transposomes.

PubMed

Chen, He; Yao, Jiacheng; Fu, Yusi; Pang, Yuhong; Wang, Jianbin; Huang, Yanyi

2018-04-11

The next generation sequencing (NGS) technologies have been rapidly evolved and applied to various research fields, but they often suffer from losing long-range information due to short library size and read length. Here, we develop a simple, cost-efficient, and versatile NGS library preparation method, called tagmentation on microbeads (TOM). This method is capable of recovering long-range information through tagmentation mediated by microbead-immobilized transposomes. Using transposomes with DNA barcodes to identically label adjacent sequences during tagmentation, we can restore inter-read connection of each fragment from original DNA molecule by fragment-barcode linkage after sequencing. In our proof-of-principle experiment, more than 4.5% of the reads are linked with their adjacent reads, and the longest linkage is over 1112 bp. We demonstrate TOM with eight barcodes, but the number of barcodes can be scaled up by an ultrahigh complexity construction. We also show this method has low amplification bias and effectively fits the applications to identify copy number variations.

Scaling up the 454 Titanium Library Construction and Pooling of Barcoded Libraries

DOE Office of Scientific and Technical Information (OSTI.GOV)

Phung, Wilson; Hack, Christopher; Shapiro, Harris

2009-03-23

We have been developing a high throughput 454 library construction process at the Joint Genome Institute to meet the needs of de novo sequencing a large number of microbial and eukaryote genomes, EST, and metagenome projects. We have been focusing efforts in three areas: (1) modifying the current process to allow the construction of 454 standard libraries on a 96-well format; (2) developing a robotic platform to perform the 454 library construction; and (3) designing molecular barcodes to allow pooling and sorting of many different samples. In the development of a high throughput process to scale up the number ofmore » libraries by adapting the process to a 96-well plate format, the key process change involves the replacement of gel electrophoresis for size selection with Solid Phase Reversible Immobilization (SPRI) beads. Although the standard deviation of the insert sizes increases, the overall quality sequence and distribution of the reads in the genome has not changed. The manual process of constructing 454 shotgun libraries on 96-well plates is a time-consuming, labor-intensive, and ergonomically hazardous process; we have been experimenting to program a BioMek robot to perform the library construction. This will not only enable library construction to be completed in a single day, but will also minimize any ergonomic risk. In addition, we have implemented a set of molecular barcodes (AKA Multiple Identifiers or MID) and a pooling process that allows us to sequence many targets simultaneously. Here we will present the testing of pooling a set of selected fosmids derived from the endomycorrhizal fungus Glomus intraradices. By combining the robotic library construction process and the use of molecular barcodes, it is now possible to sequence hundreds of fosmids that represent a minimal tiling path of this genome. Here we present the progress and the challenges of developing these scaled-up processes.« less

First large-scale DNA barcoding assessment of reptiles in the biodiversity hotspot of Madagascar, based on newly designed COI primers.

PubMed

Nagy, Zoltán T; Sonet, Gontran; Glaw, Frank; Vences, Miguel

2012-01-01

DNA barcoding of non-avian reptiles based on the cytochrome oxidase subunit I (COI) gene is still in a very early stage, mainly due to technical problems. Using a newly developed set of reptile-specific primers for COI we present the first comprehensive study targeting the entire reptile fauna of the fourth-largest island in the world, the biodiversity hotspot of Madagascar. Representatives of the majority of Madagascan non-avian reptile species (including Squamata and Testudines) were sampled and successfully DNA barcoded. The new primer pair achieved a constantly high success rate (72.7-100%) for most squamates. More than 250 species of reptiles (out of the 393 described ones; representing around 64% of the known diversity of species) were barcoded. The average interspecific genetic distance within families ranged from a low of 13.4% in the Boidae to a high of 29.8% in the Gekkonidae. Using the average genetic divergence between sister species as a threshold, 41-48 new candidate (undescribed) species were identified. Simulations were used to evaluate the performance of DNA barcoding as a function of completeness of taxon sampling and fragment length. Compared with available multi-gene phylogenies, DNA barcoding correctly assigned most samples to species, genus and family with high confidence and the analysis of fewer taxa resulted in an increased number of well supported lineages. Shorter marker-lengths generally decreased the number of well supported nodes, but even mini-barcodes of 100 bp correctly assigned many samples to genus and family. The new protocols might help to promote DNA barcoding of reptiles and the established library of reference DNA barcodes will facilitate the molecular identification of Madagascan reptiles. Our results might be useful to easily recognize undescribed diversity (i.e. novel taxa), to resolve taxonomic problems, and to monitor the international pet trade without specialized expert knowledge.

First Large-Scale DNA Barcoding Assessment of Reptiles in the Biodiversity Hotspot of Madagascar, Based on Newly Designed COI Primers

PubMed Central

Nagy, Zoltán T.; Sonet, Gontran; Glaw, Frank; Vences, Miguel

2012-01-01

Background DNA barcoding of non-avian reptiles based on the cytochrome oxidase subunit I (COI) gene is still in a very early stage, mainly due to technical problems. Using a newly developed set of reptile-specific primers for COI we present the first comprehensive study targeting the entire reptile fauna of the fourth-largest island in the world, the biodiversity hotspot of Madagascar. Methodology/Principal Findings Representatives of the majority of Madagascan non-avian reptile species (including Squamata and Testudines) were sampled and successfully DNA barcoded. The new primer pair achieved a constantly high success rate (72.7–100%) for most squamates. More than 250 species of reptiles (out of the 393 described ones; representing around 64% of the known diversity of species) were barcoded. The average interspecific genetic distance within families ranged from a low of 13.4% in the Boidae to a high of 29.8% in the Gekkonidae. Using the average genetic divergence between sister species as a threshold, 41–48 new candidate (undescribed) species were identified. Simulations were used to evaluate the performance of DNA barcoding as a function of completeness of taxon sampling and fragment length. Compared with available multi-gene phylogenies, DNA barcoding correctly assigned most samples to species, genus and family with high confidence and the analysis of fewer taxa resulted in an increased number of well supported lineages. Shorter marker-lengths generally decreased the number of well supported nodes, but even mini-barcodes of 100 bp correctly assigned many samples to genus and family. Conclusions/Significance The new protocols might help to promote DNA barcoding of reptiles and the established library of reference DNA barcodes will facilitate the molecular identification of Madagascan reptiles. Our results might be useful to easily recognize undescribed diversity (i.e. novel taxa), to resolve taxonomic problems, and to monitor the international pet trade without specialized expert knowledge. PMID:22479636

A DNA barcode library for ground beetles (Insecta, Coleoptera, Carabidae) of Germany: The genus Bembidion Latreille, 1802 and allied taxa

PubMed Central

Raupach, Michael J.; Hannig, Karsten; Morinière, Jérome; Hendrich, Lars

2016-01-01

Abstract As molecular identification method, DNA barcoding based on partial cytochrome c oxidase subunit 1 (COI) sequences has been proven to be a useful tool for species determination in many insect taxa including ground beetles. In this study we tested the effectiveness of DNA barcodes to discriminate species of the ground beetle genus Bembidion and some closely related taxa of Germany. DNA barcodes were obtained from 819 individuals and 78 species, including sequences from previous studies as well as more than 300 new generated DNA barcodes. We found a 1:1 correspondence between BIN and traditionally recognized species for 69 species (89%). Low interspecific distances with maximum pairwise K2P values below 2.2% were found for three species pairs, including two species pairs with haplotype sharing (Bembidion atrocaeruleum/Bembidion varicolor and Bembidion guttula/Bembidion mannerheimii). In contrast to this, deep intraspecific sequence divergences with distinct lineages were revealed for two species (Bembidion geniculatum/Ocys harpaloides). Our study emphasizes the use of DNA barcodes for the identification of the analyzed ground beetles species and represents an important step in building-up a comprehensive barcode library for the Carabidae in Germany and Central Europe as well. PMID:27408547

Starting a DNA barcode reference library for shallow water polychaetes from the southern European Atlantic coast.

PubMed

Lobo, Jorge; Teixeira, Marcos A L; Borges, Luisa M S; Ferreira, Maria S G; Hollatz, Claudia; Gomes, Pedro T; Sousa, Ronaldo; Ravara, Ascensão; Costa, Maria H; Costa, Filipe O

2016-01-01

Annelid polychaetes have been seldom the focus of dedicated DNA barcoding studies, despite their ecological relevance and often dominance, particularly in soft-bottom estuarine and coastal marine ecosystems. Here, we report the first assessment of the performance of DNA barcodes in the discrimination of shallow water polychaete species from the southern European Atlantic coast, focusing on specimens collected in estuaries and coastal ecosystems of Portugal. We analysed cytochrome oxidase I DNA barcodes (COI-5P) from 164 specimens, which were assigned to 51 morphospecies. To our data set from Portugal, we added available published sequences selected from the same species, genus or family, to inspect for taxonomic congruence among studies and collection location. The final data set comprised 290 specimens and 79 morphospecies, which generated 99 Barcode Index Numbers (BINs) within Barcode of Life Data Systems (BOLD). Among these, 22 BINs were singletons, 47 other BINs were concordant, confirming the initial identification based on morphological characters, and 30 were discordant, most of which consisted on multiple BINs found for the same morphospecies. Some of the most prominent cases in the latter category include Hediste diversicolor (O.F. Müller, 1776) (7), Eulalia viridis (Linnaeus, 1767) (2) and Owenia fusiformis (delle Chiaje, 1844) (5), all of them reported from Portugal and frequently used in ecological studies as environmental quality indicators. Our results for these species showed discordance between molecular lineages and morphospecies, or added additional relatively divergent lineages. The potential inaccuracies in environmental assessments, where underpinning polychaete species diversity is poorly resolved or clarified, demand additional and extensive investigation of the DNA barcode diversity in this group, in parallel with alpha taxonomy efforts. © 2015 John Wiley & Sons Ltd.

DNA Barcoding for the Identification and Authentication of Animal Species in Traditional Medicine.

PubMed

Yang, Fan; Ding, Fei; Chen, Hong; He, Mingqi; Zhu, Shixin; Ma, Xin; Jiang, Li; Li, Haifeng

2018-01-01

Animal-based traditional medicine not only plays a significant role in therapeutic practices worldwide but also provides a potential compound library for drug discovery. However, persistent hunting and illegal trade markedly threaten numerous medicinal animal species, and increasing demand further provokes the emergence of various adulterants. As the conventional methods are difficult and time-consuming to detect processed products or identify animal species with similar morphology, developing novel authentication methods for animal-based traditional medicine represents an urgent need. During the last decade, DNA barcoding offers an accurate and efficient strategy that can identify existing species and discover unknown species via analysis of sequence variation in a standardized region of DNA. Recent studies have shown that DNA barcoding as well as minibarcoding and metabarcoding is capable of identifying animal species and discriminating the authentics from the adulterants in various types of traditional medicines, including raw materials, processed products, and complex preparations. These techniques can also be used to detect the unlabelled and threatened animal species in traditional medicine. Here, we review the recent progress of DNA barcoding for the identification and authentication of animal species used in traditional medicine, which provides a reference for quality control and trade supervision of animal-based traditional medicine.

DNA Barcoding for the Identification and Authentication of Animal Species in Traditional Medicine

PubMed Central

Yang, Fan; Ding, Fei; Chen, Hong; He, Mingqi; Zhu, Shixin; Ma, Xin; Jiang, Li

2018-01-01

Animal-based traditional medicine not only plays a significant role in therapeutic practices worldwide but also provides a potential compound library for drug discovery. However, persistent hunting and illegal trade markedly threaten numerous medicinal animal species, and increasing demand further provokes the emergence of various adulterants. As the conventional methods are difficult and time-consuming to detect processed products or identify animal species with similar morphology, developing novel authentication methods for animal-based traditional medicine represents an urgent need. During the last decade, DNA barcoding offers an accurate and efficient strategy that can identify existing species and discover unknown species via analysis of sequence variation in a standardized region of DNA. Recent studies have shown that DNA barcoding as well as minibarcoding and metabarcoding is capable of identifying animal species and discriminating the authentics from the adulterants in various types of traditional medicines, including raw materials, processed products, and complex preparations. These techniques can also be used to detect the unlabelled and threatened animal species in traditional medicine. Here, we review the recent progress of DNA barcoding for the identification and authentication of animal species used in traditional medicine, which provides a reference for quality control and trade supervision of animal-based traditional medicine. PMID:29849709

Diversity in a Cold Hot-Spot: DNA-Barcoding Reveals Patterns of Evolution among Antarctic Demosponges (Class Demospongiae, Phylum Porifera).

PubMed

Vargas, Sergio; Kelly, Michelle; Schnabel, Kareen; Mills, Sadie; Bowden, David; Wörheide, Gert

2015-01-01

The approximately 350 demosponge species that have been described from Antarctica represent a faunistic component distinct from that of neighboring regions. Sponges provide structure to the Antarctic benthos and refuge to other invertebrates, and can be dominant in some communities. Despite the importance of sponges in the Antarctic subtidal environment, sponge DNA barcodes are scarce but can provide insight into the evolutionary relationships of this unique biogeographic province. We sequenced the standard barcoding COI region for a comprehensive selection of sponges collected during expeditions to the Ross Sea region in 2004 and 2008, and produced DNA-barcodes for 53 demosponge species covering about 60% of the species collected. The Antarctic sponge communities are phylogenetically diverse, matching the diversity of well-sampled sponge communities in the Lusitanic and Mediterranean marine provinces in the Temperate Northern Atlantic for which molecular data are readily available. Additionally, DNA-barcoding revealed levels of in situ molecular evolution comparable to those present among Caribbean sponges. DNA-barcoding using the Segregating Sites Algorithm correctly assigned approximately 54% of the barcoded species to the morphologically determined species. A barcode library for Antarctic sponges was assembled and used to advance the systematic and evolutionary research of Antarctic sponges. We provide insights on the evolutionary forces shaping Antarctica's diverse sponge communities, and a barcode library against which future sequence data from other regions or depth strata of Antarctica can be compared. The opportunity for rapid taxonomic identification of sponge collections for ecological research is now at the horizon.

Je, a versatile suite to handle multiplexed NGS libraries with unique molecular identifiers.

PubMed

Girardot, Charles; Scholtalbers, Jelle; Sauer, Sajoscha; Su, Shu-Yi; Furlong, Eileen E M

2016-10-08

The yield obtained from next generation sequencers has increased almost exponentially in recent years, making sample multiplexing common practice. While barcodes (known sequences of fixed length) primarily encode the sample identity of sequenced DNA fragments, barcodes made of random sequences (Unique Molecular Identifier or UMIs) are often used to distinguish between PCR duplicates and transcript abundance in, for example, single-cell RNA sequencing (scRNA-seq). In paired-end sequencing, different barcodes can be inserted at each fragment end to either increase the number of multiplexed samples in the library or to use one of the barcodes as UMI. Alternatively, UMIs can be combined with the sample barcodes into composite barcodes, or with standard Illumina® indexing. Subsequent analysis must take read duplicates and sample identity into account, by identifying UMIs. Existing tools do not support these complex barcoding configurations and custom code development is frequently required. Here, we present Je, a suite of tools that accommodates complex barcoding strategies, extracts UMIs and filters read duplicates taking UMIs into account. Using Je on publicly available scRNA-seq and iCLIP data containing UMIs, the number of unique reads increased by up to 36 %, compared to when UMIs are ignored. Je is implemented in JAVA and uses the Picard API. Code, executables and documentation are freely available at http://gbcs.embl.de/Je . Je can also be easily installed in Galaxy through the Galaxy toolshed.

Revealing Hidden Diversity of the Underestimated Neotropical Ichthyofauna: DNA Barcoding in the Recently Described Genus Megaleporinus (Characiformes: Anostomidae)

PubMed Central

Ramirez, Jorge L.; Birindelli, Jose L.; Carvalho, Daniel C.; Affonso, Paulo R. A. M.; Venere, Paulo C.; Ortega, Hernán; Carrillo-Avila, Mauricio; Rodríguez-Pulido, José A.; Galetti, Pedro M.

2017-01-01

Molecular studies have improved our knowledge on the neotropical ichthyofauna. DNA barcoding has successfully been used in fish species identification and in detecting cryptic diversity. Megaleporinus (Anostomidae) is a recently described freshwater fish genus within which taxonomic uncertainties remain. Here we assessed all nominal species of this genus using a DNA barcode approach (Cytochrome Oxidase subunit I) with a broad sampling to generate a reference library, characterize new molecular lineages, and test the hypothesis that some of the nominal species represent species complexes. The analyses identified 16 (ABGD and BIN) to 18 (ABGD, GMYC, and PTP) different molecular operational taxonomic units (MOTUs) within the 10 studied nominal species, indicating cryptic biodiversity and potential candidate species. Only Megaleporinus brinco, Megaleporinus garmani, and Megaleporinus elongatus showed correspondence between nominal species and MOTUs. Within six nominal species, a subdivision in two MOTUs was found, while Megaleporinus obtusidens was divided in three MOTUs, suggesting that DNA barcode is a very useful approach to identify the molecular lineages of Megaleporinus, even in the case of recent divergence (< 0.5 Ma). Our results thus provided molecular findings that can be used along with morphological traits to better define each species, including candidate new species. This is the most complete analysis of DNA barcode in this recently described genus, and considering its economic value, a precise species identification is quite desirable and fundamental for conservation of the whole biodiversity of this fish. PMID:29075287

DNA Barcoding the Geometrid Fauna of Bavaria (Lepidoptera): Successes, Surprises, and Questions

PubMed Central

Hausmann, Axel; Haszprunar, Gerhard; Hebert, Paul D. N.

2011-01-01

Background The State of Bavaria is involved in a research program that will lead to the construction of a DNA barcode library for all animal species within its territorial boundaries. The present study provides a comprehensive DNA barcode library for the Geometridae, one of the most diverse of insect families. Methodology/Principal Findings This study reports DNA barcodes for 400 Bavarian geometrid species, 98 per cent of the known fauna, and approximately one per cent of all Bavarian animal species. Although 98.5% of these species possess diagnostic barcode sequences in Bavaria, records from neighbouring countries suggest that species-level resolution may be compromised in up to 3.5% of cases. All taxa which apparently share barcodes are discussed in detail. One case of modest divergence (1.4%) revealed a species overlooked by the current taxonomic system: Eupithecia goossensiata Mabille, 1869 stat.n. is raised from synonymy with Eupithecia absinthiata (Clerck, 1759) to species rank. Deep intraspecific sequence divergences (>2%) were detected in 20 traditionally recognized species. Conclusions/Significance The study emphasizes the effectiveness of DNA barcoding as a tool for monitoring biodiversity. Open access is provided to a data set that includes records for 1,395 geometrid specimens (331 species) from Bavaria, with 69 additional species from neighbouring regions. Taxa with deep intraspecific sequence divergences are undergoing more detailed analysis to ascertain if they represent cases of cryptic diversity. PMID:21423340

Preparation of next-generation sequencing libraries using Nextera™ technology: simultaneous DNA fragmentation and adaptor tagging by in vitro transposition.

PubMed

Caruccio, Nicholas

2011-01-01

DNA library preparation is a common entry point and bottleneck for next-generation sequencing. Current methods generally consist of distinct steps that often involve significant sample loss and hands-on time: DNA fragmentation, end-polishing, and adaptor-ligation. In vitro transposition with Nextera™ Transposomes simultaneously fragments and covalently tags the target DNA, thereby combining these three distinct steps into a single reaction. Platform-specific sequencing adaptors can be added, and the sample can be enriched and bar-coded using limited-cycle PCR to prepare di-tagged DNA fragment libraries. Nextera technology offers a streamlined, efficient, and high-throughput method for generating bar-coded libraries compatible with multiple next-generation sequencing platforms.

DNA barcoding reveal patterns of species diversity among northwestern Pacific molluscs

PubMed Central

Sun, Shao’e; Li, Qi; Kong, Lingfeng; Yu, Hong; Zheng, Xiaodong; Yu, Ruihai; Dai, Lina; Sun, Yan; Chen, Jun; Liu, Jun; Ni, Lehai; Feng, Yanwei; Yu, Zhenzhen; Zou, Shanmei; Lin, Jiping

2016-01-01

This study represents the first comprehensive molecular assessment of northwestern Pacific molluscs. In total, 2801 DNA barcodes belonging to 569 species from China, Japan and Korea were analyzed. An overlap between intra- and interspecific genetic distances was present in 71 species. We tested the efficacy of this library by simulating a sequence-based specimen identification scenario using Best Match (BM), Best Close Match (BCM) and All Species Barcode (ASB) criteria with three threshold values. BM approach returned 89.15% true identifications (95.27% when excluding singletons). The highest success rate of congruent identifications was obtained with BCM at 0.053 threshold. The analysis of our barcode library together with public data resulted in 582 Barcode Index Numbers (BINs), 72.2% of which was found to be concordantly with morphology-based identifications. The discrepancies were divided in two groups: sequences from different species clustered in a single BIN and conspecific sequences divided in one more BINs. In Neighbour-Joining phenogram, 2,320 (83.0%) queries fromed 355 (62.4%) species-specific barcode clusters allowing their successful identification. 33 species showed paraphyletic and haplotype sharing. 62 cases are represented by deeply diverged lineages. This study suggest an increased species diversity in this region, highlighting taxonomic revision and conservation strategy for the cryptic complexes. PMID:27640675

Analyzing Mosquito (Diptera: Culicidae) Diversity in Pakistan by DNA Barcoding

PubMed Central

Ashfaq, Muhammad; Hebert, Paul D. N.; Mirza, Jawwad H.; Khan, Arif M.; Zafar, Yusuf; Mirza, M. Sajjad

2014-01-01

Background Although they are important disease vectors mosquito biodiversity in Pakistan is poorly known. Recent epidemics of dengue fever have revealed the need for more detailed understanding of the diversity and distributions of mosquito species in this region. DNA barcoding improves the accuracy of mosquito inventories because morphological differences between many species are subtle, leading to misidentifications. Methodology/Principal Findings Sequence variation in the barcode region of the mitochondrial COI gene was used to identify mosquito species, reveal genetic diversity, and map the distribution of the dengue-vector species in Pakistan. Analysis of 1684 mosquitoes from 491 sites in Punjab and Khyber Pakhtunkhwa during 2010–2013 revealed 32 species with the assemblage dominated by Culex quinquefasciatus (61% of the collection). The genus Aedes (Stegomyia) comprised 15% of the specimens, and was represented by six taxa with the two dengue vector species, Ae. albopictus and Ae. aegypti, dominant and broadly distributed. Anopheles made up another 6% of the catch with An. subpictus dominating. Barcode sequence divergence in conspecific specimens ranged from 0–2.4%, while congeneric species showed from 2.3–17.8% divergence. A global haplotype analysis of disease-vectors showed the presence of multiple haplotypes, although a single haplotype of each dengue-vector species was dominant in most countries. Geographic distribution of Ae. aegypti and Ae. albopictus showed the later species was dominant and found in both rural and urban environments. Conclusions As the first DNA-based analysis of mosquitoes in Pakistan, this study has begun the construction of a barcode reference library for the mosquitoes of this region. Levels of genetic diversity varied among species. Because of its capacity to differentiate species, even those with subtle morphological differences, DNA barcoding aids accurate tracking of vector populations. PMID:24827460

Analyzing mosquito (Diptera: culicidae) diversity in Pakistan by DNA barcoding.

PubMed

Ashfaq, Muhammad; Hebert, Paul D N; Mirza, Jawwad H; Khan, Arif M; Zafar, Yusuf; Mirza, M Sajjad

2014-01-01

Although they are important disease vectors mosquito biodiversity in Pakistan is poorly known. Recent epidemics of dengue fever have revealed the need for more detailed understanding of the diversity and distributions of mosquito species in this region. DNA barcoding improves the accuracy of mosquito inventories because morphological differences between many species are subtle, leading to misidentifications. Sequence variation in the barcode region of the mitochondrial COI gene was used to identify mosquito species, reveal genetic diversity, and map the distribution of the dengue-vector species in Pakistan. Analysis of 1684 mosquitoes from 491 sites in Punjab and Khyber Pakhtunkhwa during 2010-2013 revealed 32 species with the assemblage dominated by Culex quinquefasciatus (61% of the collection). The genus Aedes (Stegomyia) comprised 15% of the specimens, and was represented by six taxa with the two dengue vector species, Ae. albopictus and Ae. aegypti, dominant and broadly distributed. Anopheles made up another 6% of the catch with An. subpictus dominating. Barcode sequence divergence in conspecific specimens ranged from 0-2.4%, while congeneric species showed from 2.3-17.8% divergence. A global haplotype analysis of disease-vectors showed the presence of multiple haplotypes, although a single haplotype of each dengue-vector species was dominant in most countries. Geographic distribution of Ae. aegypti and Ae. albopictus showed the later species was dominant and found in both rural and urban environments. As the first DNA-based analysis of mosquitoes in Pakistan, this study has begun the construction of a barcode reference library for the mosquitoes of this region. Levels of genetic diversity varied among species. Because of its capacity to differentiate species, even those with subtle morphological differences, DNA barcoding aids accurate tracking of vector populations.

Calibrating snakehead diversity with DNA barcodes: expanding taxonomic coverage to enable identification of potential and established invasive species.

PubMed

Serrao, Natasha R; Steinke, Dirk; Hanner, Robert H

2014-01-01

Detecting and documenting the occurrence of invasive species outside their native range requires tools to support their identification. This can be challenging for taxa with diverse life stages and/or problematic or unresolved morphological taxonomies. DNA barcoding provides a potent method for identifying invasive species, as it allows for species identification at all life stages, including fragmentary remains. It also provides an efficient interim taxonomic framework for quantifying cryptic genetic diversity by parsing barcode sequences into discontinuous haplogroup clusters (typical of reproductively isolated species) and labelling them with unique alphanumeric identifiers. Snakehead fishes are a diverse group of opportunistic predators endemic to Asia and Africa that may potentially pose significant threats as aquatic invasive species. At least three snakehead species (Channa argus, C. maculata, and C. marulius) are thought to have entered North America through the aquarium and live-food fish markets, and have established populations, yet their origins remain unclear. The objectives of this study were to assemble a library of DNA barcode sequences derived from expert identified reference specimens in order to determine the identity and aid invasion pathway analysis of the non-indigenous species found in North America using DNA barcodes. Sequences were obtained from 121 tissue samples representing 25 species and combined with public records from GenBank for a total of 36 putative species, which then partitioned into 49 discrete haplogroups. Multiple divergent clusters were observed within C. gachua, C. marulius, C. punctata and C. striata suggesting the potential presence of cryptic species diversity within these lineages. Our findings demonstrate that DNA barcoding is a valuable tool for species identification in challenging and under-studied taxonomic groups such as snakeheads, and provides a useful framework for inferring invasion pathway analysis.

The diversity and biogeography of the Coleoptera of Churchill: insights from DNA barcoding

PubMed Central

2013-01-01

Background Coleoptera is the most diverse order of insects (>300,000 described species), but its richness diminishes at increasing latitudes (e.g., ca. 7400 species recorded in Canada), particularly of phytophagous and detritivorous species. However, incomplete sampling of northern habitats and a lack of taxonomic study of some families limits our understanding of biodiversity patterns in the Coleoptera. We conducted an intensive biodiversity survey from 2006–2010 at Churchill, Manitoba, Canada in order to quantify beetle species diversity in this model region, and to prepare a barcode library of beetles for sub-arctic biodiversity and ecological research. We employed DNA barcoding to provide estimates of provisional species diversity, including for families currently lacking taxonomic expertise, and to examine the guild structure, habitat distribution, and biogeography of beetles in the Churchill region. Results We obtained DNA barcodes from 3203 specimens representing 302 species or provisional species (the latter quantitatively defined on the basis of Molecular Operational Taxonomic Units, MOTUs) in 31 families of Coleoptera. Of the 184 taxa identified to the level of a Linnaean species name, 170 (92.4%) corresponded to a single MOTU, four (2.2%) represented closely related sibling species pairs within a single MOTU, and ten (5.4%) were divided into two or more MOTUs suggestive of cryptic species. The most diverse families were the Dytiscidae (63 spp.), Staphylinidae (54 spp.), and Carabidae (52 spp.), although the accumulation curve for Staphylinidae suggests that considerable additional diversity remains to be sampled in this family. Most of the species present are predatory, with phytophagous, mycophagous, and saprophagous guilds being represented by fewer species. Most named species of Carabidae and Dytiscidae showed a significant bias toward open habitats (wet or dry). Forest habitats, particularly dry boreal forest, although limited in extent in the region, were undersampled. Conclusions We present an updated species list for this region as well as a species-level DNA barcode reference library. This resource will facilitate future work, such as biomonitoring and the study of the ecology and distribution of larvae. PMID:24164967

The Application of DNA Barcodes for the Identification of Marine Crustaceans from the North Sea and Adjacent Regions.

PubMed

Raupach, Michael J; Barco, Andrea; Steinke, Dirk; Beermann, Jan; Laakmann, Silke; Mohrbeck, Inga; Neumann, Hermann; Kihara, Terue C; Pointner, Karin; Radulovici, Adriana; Segelken-Voigt, Alexandra; Wesse, Christina; Knebelsberger, Thomas

2015-01-01

During the last years DNA barcoding has become a popular method of choice for molecular specimen identification. Here we present a comprehensive DNA barcode library of various crustacean taxa found in the North Sea, one of the most extensively studied marine regions of the world. Our data set includes 1,332 barcodes covering 205 species, including taxa of the Amphipoda, Copepoda, Decapoda, Isopoda, Thecostraca, and others. This dataset represents the most extensive DNA barcode library of the Crustacea in terms of species number to date. By using the Barcode of Life Data Systems (BOLD), unique BINs were identified for 198 (96.6%) of the analyzed species. Six species were characterized by two BINs (2.9%), and three BINs were found for the amphipod species Gammarus salinus Spooner, 1947 (0.4%). Intraspecific distances with values higher than 2.2% were revealed for 13 species (6.3%). Exceptionally high distances of up to 14.87% between two distinct but monophyletic clusters were found for the parasitic copepod Caligus elongatus Nordmann, 1832, supporting the results of previous studies that indicated the existence of an overlooked sea louse species. In contrast to these high distances, haplotype-sharing was observed for two decapod spider crab species, Macropodia parva Van Noort & Adema, 1985 and Macropodia rostrata (Linnaeus, 1761), underlining the need for a taxonomic revision of both species. Summarizing the results, our study confirms the application of DNA barcodes as highly effective identification system for the analyzed marine crustaceans of the North Sea and represents an important milestone for modern biodiversity assessment studies using barcode sequences.

The neotype barcode of the cotton aphid (Hemiptera: Aphididae: Aphis gossypii Glover, 1877) and a proposal for type barcodes

USDA-ARS?s Scientific Manuscript database

A type barcode is a DNA barcode unequivocally tied to an authoritatively identified specimen, preferably the primary type specimen. Type barcodes are analogous, albeit subordinate, to type specimens, providing a stable reference to which other barcodes can be compared. We here designate and describe...

Developing a DNA barcode library for perciform fishes in the South China Sea: Species identification, accuracy and cryptic diversity.

PubMed

Hou, Gang; Chen, Wei-Tao; Lu, Huo-Sheng; Cheng, Fei; Xie, Song-Guang

2018-01-01

DNA barcodes were studied for 1,353 specimens representing 272 morphological species belonging to 149 genera and 55 families of Perciformes from the South China Sea (SCS). The average Kimura 2-parameter (K2P) distances within species, genera and families were 0.31%, 8.71% and 14.52%, respectively. A neighbour-joining (NJ) tree, Bayesian inference (BI) and maximum-likelihood (ML) trees and Automatic Barcode Gap Discovery (ABGD) revealed 260, 253 and 259 single-species-representing clusters, respectively. Barcoding gap analysis (BGA) demonstrated that barcode gaps were present for 178 of 187 species analysed with multiple specimens (95.2%), with the minimum interspecific distance to the nearest neighbour larger than the maximum intraspecific distance. A group of three Thunnus species (T. albacares, T. obesus and T. tonggol), a pair of Gerres species (G. oyena and G. japonicus), a pair of Istiblennius species (I. edentulous and I. lineatus) and a pair of Uranoscopus species (U. oligolepis and U. kaianus) were observed with low interspecific distances and overlaps between intra- and interspecific genetic distances. Three species (Apogon ellioti, Naucrates ductor and Psenopsis anomala) showed deep intraspecific divergences and generated two lineages each, suggesting the possibility of cryptic species. Our results demonstrated that DNA barcodes are highly reliable for delineating species of Perciformes in the SCS. The DNA barcode library established in this study will shed light on further research on the diversity of Perciformes in the SCS. © 2017 John Wiley & Sons Ltd.

A flexible and economical barcoding approach for highly multiplexed amplicon sequencing of diverse target genes

PubMed Central

Herbold, Craig W.; Pelikan, Claus; Kuzyk, Orest; Hausmann, Bela; Angel, Roey; Berry, David; Loy, Alexander

2015-01-01

High throughput sequencing of phylogenetic and functional gene amplicons provides tremendous insight into the structure and functional potential of complex microbial communities. Here, we introduce a highly adaptable and economical PCR approach to barcoding and pooling libraries of numerous target genes. In this approach, we replace gene- and sequencing platform-specific fusion primers with general, interchangeable barcoding primers, enabling nearly limitless customized barcode-primer combinations. Compared to barcoding with long fusion primers, our multiple-target gene approach is more economical because it overall requires lower number of primers and is based on short primers with generally lower synthesis and purification costs. To highlight our approach, we pooled over 900 different small-subunit rRNA and functional gene amplicon libraries obtained from various environmental or host-associated microbial community samples into a single, paired-end Illumina MiSeq run. Although the amplicon regions ranged in size from approximately 290 to 720 bp, we found no significant systematic sequencing bias related to amplicon length or gene target. Our results indicate that this flexible multiplexing approach produces large, diverse, and high quality sets of amplicon sequence data for modern studies in microbial ecology. PMID:26236305

Multiplex single-molecule interaction profiling of DNA-barcoded proteins.

PubMed

Gu, Liangcai; Li, Chao; Aach, John; Hill, David E; Vidal, Marc; Church, George M

2014-11-27

In contrast with advances in massively parallel DNA sequencing, high-throughput protein analyses are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule protein detection using optical methods is limited by the number of spectrally non-overlapping chromophores. Here we introduce a single-molecular-interaction sequencing (SMI-seq) technology for parallel protein interaction profiling leveraging single-molecule advantages. DNA barcodes are attached to proteins collectively via ribosome display or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide thin film to construct a random single-molecule array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies) and analysed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimetre. Furthermore, protein interactions can be measured on the basis of the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor and antibody-binding profiling, are demonstrated. SMI-seq enables 'library versus library' screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity.

The Scirtothrips dorsalis Species Complex: Endemism and Invasion in a Global Pest

PubMed Central

Dickey, Aaron M.; Kumar, Vivek; Hoddle, Mark S.; Funderburk, Joe E.; Morgan, J. Kent; Jara-Cavieres, Antonella; Shatters, Robert G. Jr.; Osborne, Lance S.; McKenzie, Cindy L.

2015-01-01

Invasive arthropods pose unique management challenges in various environments, the first of which is correct identification. This apparently mundane task is particularly difficult if multiple species are morphologically indistinguishable but accurate identification can be determined with DNA barcoding provided an adequate reference set is available. Scirtothrips dorsalis is a highly polyphagous plant pest with a rapidly expanding global distribution and this species, as currently recognized, may be comprised of cryptic species. Here we report the development of a comprehensive DNA barcode library for S. dorsalis and seven nuclear markers via next-generation sequencing for identification use within the complex. We also report the delimitation of nine cryptic species and two morphologically distinguishable species comprising the S. dorsalis species complex using histogram analysis of DNA barcodes, Bayesian phylogenetics, and the multi-species coalescent. One member of the complex, here designated the South Asia 1 cryptic species, is highly invasive, polyphagous, and likely the species implicated in tospovirus transmission. Two other species, South Asia 2, and East Asia 1 are also highly polyphagous and appear to be at an earlier stage of global invasion. The remaining members of the complex are regionally endemic, varying in their pest status and degree of polyphagy. In addition to patterns of invasion and endemism, our results provide a framework both for identifying members of the complex based on their DNA barcode, and for future species delimiting efforts. PMID:25893251

Ten years of barcoding at the African Centre for DNA Barcoding.

PubMed

Bezeng, B S; Davies, T J; Daru, B H; Kabongo, R M; Maurin, O; Yessoufou, K; van der Bank, H; van der Bank, M

2017-07-01

The African Centre for DNA Barcoding (ACDB) was established in 2005 as part of a global initiative to accurately and rapidly survey biodiversity using short DNA sequences. The mitochondrial cytochrome c oxidase 1 gene (CO1) was rapidly adopted as the de facto barcode for animals. Following the evaluation of several candidate loci for plants, the Plant Working Group of the Consortium for the Barcoding of Life in 2009 recommended that two plastid genes, rbcLa and matK, be adopted as core DNA barcodes for terrestrial plants. To date, numerous studies continue to test the discriminatory power of these markers across various plant lineages. Over the past decade, we at the ACDB have used these core DNA barcodes to generate a barcode library for southern Africa. To date, the ACDB has contributed more than 21 000 plant barcodes and over 3000 CO1 barcodes for animals to the Barcode of Life Database (BOLD). Building upon this effort, we at the ACDB have addressed questions related to community assembly, biogeography, phylogenetic diversification, and invasion biology. Collectively, our work demonstrates the diverse applications of DNA barcoding in ecology, systematics, evolutionary biology, and conservation.

DNA barcoding facilitates associations and diagnoses for Trichoptera larvae of the Churchill (Manitoba, Canada) area.

PubMed

Ruiter, David E; Boyle, Elizabeth E; Zhou, Xin

2013-02-20

The North American Trichoptera larvae are poorly known at the species level, despite their importance in the understanding of freshwater fauna and critical use in biomonitoring. This study focused on morphological diagnoses for larvae occurring in the Churchill, Manitoba area, representing the largest larval association effort for the caddisflies at any given locality thus far. The current DNA barcode reference library of Trichoptera (available on the Barcode of Life Data Systems) was utilized to provide larval-adult associations. The present study collected an additional 23 new species records for the Churchill area, increasing the total Trichoptera richness to 91 species. We were able to associate 62 larval taxa, comprising 68.1% of the Churchill area Trichoptera taxa. This endeavor to identify immature life stage for the caddisflies enabled the development of morphological diagnoses, production of photographs and an appropriate taxonomic key to facilitate larval species analyses in the area. The use of DNA for associations of unknown larvae with known adults proved rapid and successful. This method should accelerate the state-of-knowledge for North American Trichoptera larvae as well as other taxonomic lineages. The morphological analysis should be useful for determination of material from the Churchill area.

A DNA 'barcode blitz': rapid digitization and sequencing of a natural history collection.

PubMed

Hebert, Paul D N; Dewaard, Jeremy R; Zakharov, Evgeny V; Prosser, Sean W J; Sones, Jayme E; McKeown, Jaclyn T A; Mantle, Beth; La Salle, John

2013-01-01

DNA barcoding protocols require the linkage of each sequence record to a voucher specimen that has, whenever possible, been authoritatively identified. Natural history collections would seem an ideal resource for barcode library construction, but they have never seen large-scale analysis because of concerns linked to DNA degradation. The present study examines the strength of this barrier, carrying out a comprehensive analysis of moth and butterfly (Lepidoptera) species in the Australian National Insect Collection. Protocols were developed that enabled tissue samples, specimen data, and images to be assembled rapidly. Using these methods, a five-person team processed 41,650 specimens representing 12,699 species in 14 weeks. Subsequent molecular analysis took about six months, reflecting the need for multiple rounds of PCR as sequence recovery was impacted by age, body size, and collection protocols. Despite these variables and the fact that specimens averaged 30.4 years old, barcode records were obtained from 86% of the species. In fact, one or more barcode compliant sequences (>487 bp) were recovered from virtually all species represented by five or more individuals, even when the youngest was 50 years old. By assembling specimen images, distributional data, and DNA barcode sequences on a web-accessible informatics platform, this study has greatly advanced accessibility to information on thousands of species. Moreover, much of the specimen data became publically accessible within days of its acquisition, while most sequence results saw release within three months. As such, this study reveals the speed with which DNA barcode workflows can mobilize biodiversity data, often providing the first web-accessible information for a species. These results further suggest that existing collections can enable the rapid development of a comprehensive DNA barcode library for the most diverse compartment of terrestrial biodiversity - insects.

The Application of DNA Barcodes for the Identification of Marine Crustaceans from the North Sea and Adjacent Regions

PubMed Central

Raupach, Michael J.; Barco, Andrea; Steinke, Dirk; Beermann, Jan; Laakmann, Silke; Mohrbeck, Inga; Neumann, Hermann; Kihara, Terue C.; Pointner, Karin; Radulovici, Adriana; Segelken-Voigt, Alexandra; Wesse, Christina; Knebelsberger, Thomas

2015-01-01

During the last years DNA barcoding has become a popular method of choice for molecular specimen identification. Here we present a comprehensive DNA barcode library of various crustacean taxa found in the North Sea, one of the most extensively studied marine regions of the world. Our data set includes 1,332 barcodes covering 205 species, including taxa of the Amphipoda, Copepoda, Decapoda, Isopoda, Thecostraca, and others. This dataset represents the most extensive DNA barcode library of the Crustacea in terms of species number to date. By using the Barcode of Life Data Systems (BOLD), unique BINs were identified for 198 (96.6%) of the analyzed species. Six species were characterized by two BINs (2.9%), and three BINs were found for the amphipod species Gammarus salinus Spooner, 1947 (0.4%). Intraspecific distances with values higher than 2.2% were revealed for 13 species (6.3%). Exceptionally high distances of up to 14.87% between two distinct but monophyletic clusters were found for the parasitic copepod Caligus elongatus Nordmann, 1832, supporting the results of previous studies that indicated the existence of an overlooked sea louse species. In contrast to these high distances, haplotype-sharing was observed for two decapod spider crab species, Macropodia parva Van Noort & Adema, 1985 and Macropodia rostrata (Linnaeus, 1761), underlining the need for a taxonomic revision of both species. Summarizing the results, our study confirms the application of DNA barcodes as highly effective identification system for the analyzed marine crustaceans of the North Sea and represents an important milestone for modern biodiversity assessment studies using barcode sequences. PMID:26417993

Towards a global barcode library for Lymantria (Lepidoptera: Lymantriinae) tussock moths of biosecurity concern

Treesearch

Jeremy R. deWaard; Andrew Mitchell; Melody A. Keena; David Gopurenko; Laura M. Boykin; Karen F. Armstrong; Michael G. Pogue; Joao Lima; Robin Floyd; Robert H. Hanner; Leland M. Humble

2010-01-01

This study demonstrates the efficacy of DNA barcodes for diagnosing species of Lymantria and reinforces the view that the approach is an under-utilized resource with substantial potential for biosecurity and surveillance. Biomonitoring agencies currently employing the NB restriction digest system would gather more information by transitioning to the...

Colony-PCR Is a Rapid Method for DNA Amplification of Hyphomycetes

PubMed Central

Walch, Georg; Knapp, Maria; Rainer, Georg; Peintner, Ursula

2016-01-01

Fungal pure cultures identified with both classical morphological methods and through barcoding sequences are a basic requirement for reliable reference sequences in public databases. Improved techniques for an accelerated DNA barcode reference library construction will result in considerably improved sequence databases covering a wider taxonomic range. Fast, cheap, and reliable methods for obtaining DNA sequences from fungal isolates are, therefore, a valuable tool for the scientific community. Direct colony PCR was already successfully established for yeasts, but has not been evaluated for a wide range of anamorphic soil fungi up to now, and a direct amplification protocol for hyphomycetes without tissue pre-treatment has not been published so far. Here, we present a colony PCR technique directly from fungal hyphae without previous DNA extraction or other prior manipulation. Seven hundred eighty-eight fungal strains from 48 genera were tested with a success rate of 86%. PCR success varied considerably: DNA of fungi belonging to the genera Cladosporium, Geomyces, Fusarium, and Mortierella could be amplified with high success. DNA of soil-borne yeasts was always successfully amplified. Absidia, Mucor, Trichoderma, and Penicillium isolates had noticeably lower PCR success. PMID:29376929

Barcoding snakeheads (Teleostei, Channidae) revisited: Discovering greater species diversity and resolving perpetuated taxonomic confusions

PubMed Central

Conte-Grand, Cecilia; Britz, Ralf; Dahanukar, Neelesh; Raghavan, Rajeev; Pethiyagoda, Rohan; Tan, Heok Hui; Hadiaty, Renny K.; Yaakob, Norsham S.

2017-01-01

Snakehead fishes of the family Channidae are predatory freshwater teleosts from Africa and Asia comprising 38 valid species. Snakeheads are important food fishes (aquaculture, live food trade) and have been introduced widely with several species becoming highly invasive. A channid barcode library was recently assembled by Serrao and co-workers to better detect and identify potential and established invasive snakehead species outside their native range. Comparing our own recent phylogenetic results of this taxonomically confusing group with those previously reported revealed several inconsistencies that prompted us to expand and improve on previous studies. By generating 343 novel snakehead coxI sequences and combining them with an additional 434 coxI sequences from GenBank we highlight several problems with previous efforts towards the assembly of a snakehead reference barcode library. We found that 16.3% of the channid coxI sequences deposited in GenBank are based on misidentifications. With the inclusion of our own data we were, however, able to solve these cases of perpetuated taxonomic confusion. Different species delimitation approaches we employed (BIN, GMYC, and PTP) were congruent in suggesting a potentially much higher species diversity within snakeheads than currently recognized. In total, 90 BINs were recovered and within a total of 15 currently recognized species multiple BINs were identified. This higher species diversity is mostly due to either the incorporation of undescribed, narrow range, endemics from the Eastern Himalaya biodiversity hotspot or the incorporation of several widespread species characterized by deep genetic splits between geographically well-defined lineages. In the latter case, over-lumping in the past has deflated the actual species numbers. Further integrative approaches are clearly needed for providing a better taxonomic understanding of snakehead diversity, new species descriptions and taxonomic revisions of the group. PMID:28931084

First record and five new species of Xylographellini (Coleoptera: Ciidae) from China, with online DNA barcode library of the family.

PubMed

Lopes-Andrade, Cristiano; Grebennikov, Vasily V

2015-08-25

We report the first record of the beetle tribe Xylographellini (Ciidae) from the continental Palaearctic Region, represented by five new species discovered in Yunnan and Sichuan provinces, China: Scolytocis danae sp. nov., Syncosmetus euryale sp. nov., Sync. medusa sp. nov., Sync. perseus sp. nov. and Sync. stheno sp. nov. Illustrations and identification keys are provided for these new species, and in order to facilitate further research of Ciidae we present an open-access DNA barcode library (dx.doi.org/10.5883/DS-SYNCOSM) containing 114 records (of 44 species in 14 genera), 15 of which belong to the newly described species. A phylogenetic analysis based on the barcode fragment of the cytochrome oxidase I gene did not recover much tree structure within Ciidae, however both Xylographus Mellié and Syncosmetus Sharp were recovered as clades, with a single Scolytocis Blair being the sister to the latter.

Pair-barcode high-throughput sequencing for large-scale multiplexed sample analysis

PubMed Central

2012-01-01

Background The multiplexing becomes the major limitation of the next-generation sequencing (NGS) in application to low complexity samples. Physical space segregation allows limited multiplexing, while the existing barcode approach only permits simultaneously analysis of up to several dozen samples. Results Here we introduce pair-barcode sequencing (PBS), an economic and flexible barcoding technique that permits parallel analysis of large-scale multiplexed samples. In two pilot runs using SOLiD sequencer (Applied Biosystems Inc.), 32 independent pair-barcoded miRNA libraries were simultaneously discovered by the combination of 4 unique forward barcodes and 8 unique reverse barcodes. Over 174,000,000 reads were generated and about 64% of them are assigned to both of the barcodes. After mapping all reads to pre-miRNAs in miRBase, different miRNA expression patterns are captured from the two clinical groups. The strong correlation using different barcode pairs and the high consistency of miRNA expression in two independent runs demonstrates that PBS approach is valid. Conclusions By employing PBS approach in NGS, large-scale multiplexed pooled samples could be practically analyzed in parallel so that high-throughput sequencing economically meets the requirements of samples which are low sequencing throughput demand. PMID:22276739

Pair-barcode high-throughput sequencing for large-scale multiplexed sample analysis.

PubMed

Tu, Jing; Ge, Qinyu; Wang, Shengqin; Wang, Lei; Sun, Beili; Yang, Qi; Bai, Yunfei; Lu, Zuhong

2012-01-25

The multiplexing becomes the major limitation of the next-generation sequencing (NGS) in application to low complexity samples. Physical space segregation allows limited multiplexing, while the existing barcode approach only permits simultaneously analysis of up to several dozen samples. Here we introduce pair-barcode sequencing (PBS), an economic and flexible barcoding technique that permits parallel analysis of large-scale multiplexed samples. In two pilot runs using SOLiD sequencer (Applied Biosystems Inc.), 32 independent pair-barcoded miRNA libraries were simultaneously discovered by the combination of 4 unique forward barcodes and 8 unique reverse barcodes. Over 174,000,000 reads were generated and about 64% of them are assigned to both of the barcodes. After mapping all reads to pre-miRNAs in miRBase, different miRNA expression patterns are captured from the two clinical groups. The strong correlation using different barcode pairs and the high consistency of miRNA expression in two independent runs demonstrates that PBS approach is valid. By employing PBS approach in NGS, large-scale multiplexed pooled samples could be practically analyzed in parallel so that high-throughput sequencing economically meets the requirements of samples which are low sequencing throughput demand.

Building-up of a DNA barcode library for true bugs (insecta: hemiptera: heteroptera) of Germany reveals taxonomic uncertainties and surprises.

PubMed

Raupach, Michael J; Hendrich, Lars; Küchler, Stefan M; Deister, Fabian; Morinière, Jérome; Gossner, Martin M

2014-01-01

During the last few years, DNA barcoding has become an efficient method for the identification of species. In the case of insects, most published DNA barcoding studies focus on species of the Ephemeroptera, Trichoptera, Hymenoptera and especially Lepidoptera. In this study we test the efficiency of DNA barcoding for true bugs (Hemiptera: Heteroptera), an ecological and economical highly important as well as morphologically diverse insect taxon. As part of our study we analyzed DNA barcodes for 1742 specimens of 457 species, comprising 39 families of the Heteroptera. We found low nucleotide distances with a minimum pairwise K2P distance <2.2% within 21 species pairs (39 species). For ten of these species pairs (18 species), minimum pairwise distances were zero. In contrast to this, deep intraspecific sequence divergences with maximum pairwise distances >2.2% were detected for 16 traditionally recognized and valid species. With a successful identification rate of 91.5% (418 species) our study emphasizes the use of DNA barcodes for the identification of true bugs and represents an important step in building-up a comprehensive barcode library for true bugs in Germany and Central Europe as well. Our study also highlights the urgent necessity of taxonomic revisions for various taxa of the Heteroptera, with a special focus on various species of the Miridae. In this context we found evidence for on-going hybridization events within various taxonomically challenging genera (e.g. Nabis Latreille, 1802 (Nabidae), Lygus Hahn, 1833 (Miridae), Phytocoris Fallén, 1814 (Miridae)) as well as the putative existence of cryptic species (e.g. Aneurus avenius (Duffour, 1833) (Aradidae) or Orius niger (Wolff, 1811) (Anthocoridae)).

Building-Up of a DNA Barcode Library for True Bugs (Insecta: Hemiptera: Heteroptera) of Germany Reveals Taxonomic Uncertainties and Surprises

PubMed Central

Raupach, Michael J.; Hendrich, Lars; Küchler, Stefan M.; Deister, Fabian; Morinière, Jérome; Gossner, Martin M.

2014-01-01

During the last few years, DNA barcoding has become an efficient method for the identification of species. In the case of insects, most published DNA barcoding studies focus on species of the Ephemeroptera, Trichoptera, Hymenoptera and especially Lepidoptera. In this study we test the efficiency of DNA barcoding for true bugs (Hemiptera: Heteroptera), an ecological and economical highly important as well as morphologically diverse insect taxon. As part of our study we analyzed DNA barcodes for 1742 specimens of 457 species, comprising 39 families of the Heteroptera. We found low nucleotide distances with a minimum pairwise K2P distance <2.2% within 21 species pairs (39 species). For ten of these species pairs (18 species), minimum pairwise distances were zero. In contrast to this, deep intraspecific sequence divergences with maximum pairwise distances >2.2% were detected for 16 traditionally recognized and valid species. With a successful identification rate of 91.5% (418 species) our study emphasizes the use of DNA barcodes for the identification of true bugs and represents an important step in building-up a comprehensive barcode library for true bugs in Germany and Central Europe as well. Our study also highlights the urgent necessity of taxonomic revisions for various taxa of the Heteroptera, with a special focus on various species of the Miridae. In this context we found evidence for on-going hybridization events within various taxonomically challenging genera (e.g. Nabis Latreille, 1802 (Nabidae), Lygus Hahn, 1833 (Miridae), Phytocoris Fallén, 1814 (Miridae)) as well as the putative existence of cryptic species (e.g. Aneurus avenius (Duffour, 1833) (Aradidae) or Orius niger (Wolff, 1811) (Anthocoridae)). PMID:25203616

Spiders (Araneae) of Churchill, Manitoba: DNA barcodes and morphology reveal high species diversity and new Canadian records.

PubMed

Blagoev, Gergin A; Nikolova, Nadya I; Sobel, Crystal N; Hebert, Paul D N; Adamowicz, Sarah J

2013-11-26

Arctic ecosystems, especially those near transition zones, are expected to be strongly impacted by climate change. Because it is positioned on the ecotone between tundra and boreal forest, the Churchill area is a strategic locality for the analysis of shifts in faunal composition. This fact has motivated the effort to develop a comprehensive biodiversity inventory for the Churchill region by coupling DNA barcoding with morphological studies. The present study represents one element of this effort; it focuses on analysis of the spider fauna at Churchill. 198 species were detected among 2704 spiders analyzed, tripling the count for the Churchill region. Estimates of overall diversity suggest that another 10-20 species await detection. Most species displayed little intraspecific sequence variation (maximum <1%) in the barcode region of the cytochrome c oxidase subunit I (COI) gene, but four species showed considerably higher values (maximum = 4.1-6.2%), suggesting cryptic species. All recognized species possessed a distinct haplotype array at COI with nearest-neighbour interspecific distances averaging 8.57%. Three species new to Canada were detected: Robertus lyrifer (Theridiidae), Baryphyma trifrons (Linyphiidae), and Satilatlas monticola (Linyphiidae). The first two species may represent human-mediated introductions linked to the port in Churchill, but the other species represents a range extension from the USA. The first description of the female of S. monticola was also presented. As well, one probable new species of Alopecosa (Lycosidae) was recognized. This study provides the first comprehensive DNA barcode reference library for the spider fauna of any region. Few cryptic species of spiders were detected, a result contrasting with the prevalence of undescribed species in several other terrestrial arthropod groups at Churchill. Because most (97.5%) sequence clusters at COI corresponded with a named taxon, DNA barcoding reliably identifies spiders in the Churchill fauna. The capacity of DNA barcoding to enable the identification of otherwise taxonomically ambiguous specimens (juveniles, females) also represents a major advance for future monitoring efforts on this group.

Two new computational methods for universal DNA barcoding: a benchmark using barcode sequences of bacteria, archaea, animals, fungi, and land plants.

PubMed

Tanabe, Akifumi S; Toju, Hirokazu

2013-01-01

Taxonomic identification of biological specimens based on DNA sequence information (a.k.a. DNA barcoding) is becoming increasingly common in biodiversity science. Although several methods have been proposed, many of them are not universally applicable due to the need for prerequisite phylogenetic/machine-learning analyses, the need for huge computational resources, or the lack of a firm theoretical background. Here, we propose two new computational methods of DNA barcoding and show a benchmark for bacterial/archeal 16S, animal COX1, fungal internal transcribed spacer, and three plant chloroplast (rbcL, matK, and trnH-psbA) barcode loci that can be used to compare the performance of existing and new methods. The benchmark was performed under two alternative situations: query sequences were available in the corresponding reference sequence databases in one, but were not available in the other. In the former situation, the commonly used "1-nearest-neighbor" (1-NN) method, which assigns the taxonomic information of the most similar sequences in a reference database (i.e., BLAST-top-hit reference sequence) to a query, displays the highest rate and highest precision of successful taxonomic identification. However, in the latter situation, the 1-NN method produced extremely high rates of misidentification for all the barcode loci examined. In contrast, one of our new methods, the query-centric auto-k-nearest-neighbor (QCauto) method, consistently produced low rates of misidentification for all the loci examined in both situations. These results indicate that the 1-NN method is most suitable if the reference sequences of all potentially observable species are available in databases; otherwise, the QCauto method returns the most reliable identification results. The benchmark results also indicated that the taxon coverage of reference sequences is far from complete for genus or species level identification in all the barcode loci examined. Therefore, we need to accelerate the registration of reference barcode sequences to apply high-throughput DNA barcoding to genus or species level identification in biodiversity research.

Two New Computational Methods for Universal DNA Barcoding: A Benchmark Using Barcode Sequences of Bacteria, Archaea, Animals, Fungi, and Land Plants

PubMed Central

Tanabe, Akifumi S.; Toju, Hirokazu

2013-01-01

Taxonomic identification of biological specimens based on DNA sequence information (a.k.a. DNA barcoding) is becoming increasingly common in biodiversity science. Although several methods have been proposed, many of them are not universally applicable due to the need for prerequisite phylogenetic/machine-learning analyses, the need for huge computational resources, or the lack of a firm theoretical background. Here, we propose two new computational methods of DNA barcoding and show a benchmark for bacterial/archeal 16S, animal COX1, fungal internal transcribed spacer, and three plant chloroplast (rbcL, matK, and trnH-psbA) barcode loci that can be used to compare the performance of existing and new methods. The benchmark was performed under two alternative situations: query sequences were available in the corresponding reference sequence databases in one, but were not available in the other. In the former situation, the commonly used “1-nearest-neighbor” (1-NN) method, which assigns the taxonomic information of the most similar sequences in a reference database (i.e., BLAST-top-hit reference sequence) to a query, displays the highest rate and highest precision of successful taxonomic identification. However, in the latter situation, the 1-NN method produced extremely high rates of misidentification for all the barcode loci examined. In contrast, one of our new methods, the query-centric auto-k-nearest-neighbor (QCauto) method, consistently produced low rates of misidentification for all the loci examined in both situations. These results indicate that the 1-NN method is most suitable if the reference sequences of all potentially observable species are available in databases; otherwise, the QCauto method returns the most reliable identification results. The benchmark results also indicated that the taxon coverage of reference sequences is far from complete for genus or species level identification in all the barcode loci examined. Therefore, we need to accelerate the registration of reference barcode sequences to apply high-throughput DNA barcoding to genus or species level identification in biodiversity research. PMID:24204702

DNA barcode for the identification of the sand fly Lutzomyia longipalpis plant feeding preferences in a tropical urban environment.

PubMed

Lima, Leonardo H G de M; Mesquita, Marcelo R; Skrip, Laura; de Souza Freitas, Moisés T; Silva, Vladimir C; Kirstein, Oscar D; Abassi, Ibrahim; Warburg, Alon; Balbino, Valdir de Q; Costa, Carlos H N

2016-07-20

Little is known about the feeding behavior of hematophagous insects that require plant sugar to complete their life cycles. We studied plant feeding of Lutzomyia longipalpis sand flies, known vectors of Leishmania infantum/chagasi parasites, in a Brazilian city endemic with visceral leishmaniasis. The DNA barcode technique was applied to identify plant food source of wild-caught L. longipalpis using specific primers for a locus from the chloroplast genome, ribulose diphosphate carboxylase. DNA from all trees or shrubs within a 100-meter radius from the trap were collected to build a barcode reference library. While plants from the Anacardiaceae and Meliaceae families were the most abundant at the sampling site (25.4% and 12.7% of the local plant population, respectively), DNA from these plant families was found in few flies; in contrast, despite its low abundance (2.9%), DNA from the Fabaceae family was detected in 94.7% of the sand flies. The proportion of sand flies testing positive for DNA from a given plant family was not significantly associated with abundance, distance from the trap, or average crown expansion of plants from that family. The data suggest that there may indeed be a feeding preference of L. longipalpis for plants in the Fabaceae family.

Testing the Efficacy of DNA Barcodes for Identifying the Vascular Plants of Canada.

PubMed

Braukmann, Thomas W A; Kuzmina, Maria L; Sills, Jesse; Zakharov, Evgeny V; Hebert, Paul D N

2017-01-01

Their relatively slow rates of molecular evolution, as well as frequent exposure to hybridization and introgression, often make it difficult to discriminate species of vascular plants with the standard barcode markers (rbcL, matK, ITS2). Previous studies have examined these constraints in narrow geographic or taxonomic contexts, but the present investigation expands analysis to consider the performance of these gene regions in discriminating the species in local floras at sites across Canada. To test identification success, we employed a DNA barcode reference library with sequence records for 96% of the 5108 vascular plant species known from Canada, but coverage varied from 94% for rbcL to 60% for ITS2 and 39% for matK. Using plant lists from 27 national parks and one scientific reserve, we tested the efficacy of DNA barcodes in identifying the plants in simulated species assemblages from six biogeographic regions of Canada using BLAST and mothur. Mean pairwise distance (MPD) and mean nearest taxon distance (MNTD) were strong predictors of barcode performance for different plant families and genera, and both metrics supported ITS2 as possessing the highest genetic diversity. All three genes performed strongly in assigning the taxa present in local floras to the correct genus with values ranging from 91% for rbcL to 97% for ITS2 and 98% for matK. However, matK delivered the highest species discrimination (~81%) followed by ITS2 (~72%) and rbcL (~44%). Despite the low number of plant taxa in the Canadian Arctic, DNA barcodes had the least success in discriminating species from this biogeographic region with resolution ranging from 36% with rbcL to 69% with matK. Species resolution was higher in the other settings, peaking in the Woodland region at 52% for rbcL and 87% for matK. Our results indicate that DNA barcoding is very effective in identifying Canadian plants to a genus, and that it performs well in discriminating species in regions where floristic diversity is highest.

Testing the Efficacy of DNA Barcodes for Identifying the Vascular Plants of Canada

PubMed Central

Kuzmina, Maria L.; Sills, Jesse; Zakharov, Evgeny V.; Hebert, Paul D. N.

2017-01-01

Their relatively slow rates of molecular evolution, as well as frequent exposure to hybridization and introgression, often make it difficult to discriminate species of vascular plants with the standard barcode markers (rbcL, matK, ITS2). Previous studies have examined these constraints in narrow geographic or taxonomic contexts, but the present investigation expands analysis to consider the performance of these gene regions in discriminating the species in local floras at sites across Canada. To test identification success, we employed a DNA barcode reference library with sequence records for 96% of the 5108 vascular plant species known from Canada, but coverage varied from 94% for rbcL to 60% for ITS2 and 39% for matK. Using plant lists from 27 national parks and one scientific reserve, we tested the efficacy of DNA barcodes in identifying the plants in simulated species assemblages from six biogeographic regions of Canada using BLAST and mothur. Mean pairwise distance (MPD) and mean nearest taxon distance (MNTD) were strong predictors of barcode performance for different plant families and genera, and both metrics supported ITS2 as possessing the highest genetic diversity. All three genes performed strongly in assigning the taxa present in local floras to the correct genus with values ranging from 91% for rbcL to 97% for ITS2 and 98% for matK. However, matK delivered the highest species discrimination (~81%) followed by ITS2 (~72%) and rbcL (~44%). Despite the low number of plant taxa in the Canadian Arctic, DNA barcodes had the least success in discriminating species from this biogeographic region with resolution ranging from 36% with rbcL to 69% with matK. Species resolution was higher in the other settings, peaking in the Woodland region at 52% for rbcL and 87% for matK. Our results indicate that DNA barcoding is very effective in identifying Canadian plants to a genus, and that it performs well in discriminating species in regions where floristic diversity is highest. PMID:28072819

Multiplexing clonality: combining RGB marking and genetic barcoding

PubMed Central

Cornils, Kerstin; Thielecke, Lars; Hüser, Svenja; Forgber, Michael; Thomaschewski, Michael; Kleist, Nadja; Hussein, Kais; Riecken, Kristoffer; Volz, Tassilo; Gerdes, Sebastian; Glauche, Ingmar; Dahl, Andreas; Dandri, Maura; Roeder, Ingo; Fehse, Boris

2014-01-01

RGB marking and DNA barcoding are two cutting-edge technologies in the field of clonal cell marking. To combine the virtues of both approaches, we equipped LeGO vectors encoding red, green or blue fluorescent proteins with complex DNA barcodes carrying color-specific signatures. For these vectors, we generated highly complex plasmid libraries that were used for the production of barcoded lentiviral vector particles. In proof-of-principle experiments, we used barcoded vectors for RGB marking of cell lines and primary murine hepatocytes. We applied single-cell polymerase chain reaction to decipher barcode signatures of individual RGB-marked cells expressing defined color hues. This enabled us to prove clonal identity of cells with one and the same RGB color. Also, we made use of barcoded vectors to investigate clonal development of leukemia induced by ectopic oncogene expression in murine hematopoietic cells. In conclusion, by combining RGB marking and DNA barcoding, we have established a novel technique for the unambiguous genetic marking of individual cells in the context of normal regeneration as well as malignant outgrowth. Moreover, the introduction of color-specific signatures in barcodes will facilitate studies on the impact of different variables (e.g. vector type, transgenes, culture conditions) in the context of competitive repopulation studies. PMID:24476916

How many species and under what names? Using DNA barcoding and GenBank data for west Central African amphibian conservation

PubMed Central

Mulcahy, Daniel G.; Vanthomme, Hadrien; Tobi, Elie; Wynn, Addison H.; Zimkus, Breda M.; McDiarmid, Roy W.

2017-01-01

Development projects in west Central Africa are proceeding at an unprecedented rate, often with little concern for their effects on biodiversity. In an attempt to better understand potential impacts of a road development project on the anuran amphibian community, we conducted a biodiversity assessment employing multiple methodologies (visual encounter transects, auditory surveys, leaf litter plots and pitfall traps) to inventory species prior to construction of a new road within the buffer zone of Moukalaba-Doudou National Park, Gabon. Because of difficulties in morphological identification and taxonomic uncertainty of amphibian species observed in the area, we integrated a DNA barcoding analysis into the project to improve the overall quality and accuracy of the species inventory. Based on morphology alone, 48 species were recognized in the field and voucher specimens of each were collected. We used tissue samples from specimens collected at our field site, material available from amphibians collected in other parts of Gabon and the Republic of Congo to initiate a DNA barcode library for west Central African amphibians. We then compared our sequences with material in GenBank for the genera recorded at the study site to assist in identifications. The resulting COI and 16S barcode library allowed us to update the number of species documented at the study site to 28, thereby providing a more accurate assessment of diversity and distributions. We caution that because sequence data maintained in GenBank are often poorly curated by the original submitters and cannot be amended by third-parties, these data have limited utility for identification purposes. Nevertheless, the use of DNA barcoding is likely to benefit biodiversity inventories and long-term monitoring, particularly for taxa that can be difficult to identify based on morphology alone; likewise, inventory and monitoring programs can contribute invaluable data to the DNA barcode library and the taxonomy of complex groups. Our methods provide an example of how non-taxonomists and parataxonomists working in understudied parts of the world with limited geographic sampling and comparative morphological material can use DNA barcoding and publicly available sequence data (GenBank) to rapidly identify the number of species and assign tentative names to aid in urgent conservation management actions and contribute to taxonomic resolution. PMID:29131846

DNA barcoding of vouchered xylarium wood specimens of nine endangered Dalbergia species

Treesearch

Min Yu; Lichao Jiao; Juan Guo; Alex C. Wiedenhoeft; Tuo He; Xiaomei Jiang; Yafang Yin

2017-01-01

ITS2+trnH-psbA was the best combination of DNA barcode to resolve the Dalbergia wood species studied. We demonstrate the feasibility of building a DNA barcode reference database using xylarium wood specimens.

De novo assembly and next-generation sequencing to analyse full-length gene variants from codon-barcoded libraries.

PubMed

Cho, Namjin; Hwang, Byungjin; Yoon, Jung-ki; Park, Sangun; Lee, Joongoo; Seo, Han Na; Lee, Jeewon; Huh, Sunghoon; Chung, Jinsoo; Bang, Duhee

2015-09-21

Interpreting epistatic interactions is crucial for understanding evolutionary dynamics of complex genetic systems and unveiling structure and function of genetic pathways. Although high resolution mapping of en masse variant libraries renders molecular biologists to address genotype-phenotype relationships, long-read sequencing technology remains indispensable to assess functional relationship between mutations that lie far apart. Here, we introduce JigsawSeq for multiplexed sequence identification of pooled gene variant libraries by combining a codon-based molecular barcoding strategy and de novo assembly of short-read data. We first validate JigsawSeq on small sub-pools and observed high precision and recall at various experimental settings. With extensive simulations, we then apply JigsawSeq to large-scale gene variant libraries to show that our method can be reliably scaled using next-generation sequencing. JigsawSeq may serve as a rapid screening tool for functional genomics and offer the opportunity to explore evolutionary trajectories of protein variants.

Defining the ABC of gene essentiality in streptococci.

PubMed

Charbonneau, Amelia R L; Forman, Oliver P; Cain, Amy K; Newland, Graham; Robinson, Carl; Boursnell, Mike; Parkhill, Julian; Leigh, James A; Maskell, Duncan J; Waller, Andrew S

2017-05-31

Utilising next generation sequencing to interrogate saturated bacterial mutant libraries provides unprecedented information for the assignment of genome-wide gene essentiality. Exposure of saturated mutant libraries to specific conditions and subsequent sequencing can be exploited to uncover gene essentiality relevant to the condition. Here we present a barcoded transposon directed insertion-site sequencing (TraDIS) system to define an essential gene list for Streptococcus equi subsp. equi, the causative agent of strangles in horses, for the first time. The gene essentiality data for this group C Streptococcus was compared to that of group A and B streptococci. Six barcoded variants of pGh9:ISS1 were designed and used to generate mutant libraries containing between 33,000-66,000 unique mutants. TraDIS was performed on DNA extracted from each library and data were analysed separately and as a combined master pool. Gene essentiality determined that 19.5% of the S. equi genome was essential. Gene essentialities were compared to those of group A and group B streptococci, identifying concordances of 90.2% and 89.4%, respectively and an overall concordance of 83.7% between the three species. The use of barcoded pGh9:ISS1 to generate mutant libraries provides a highly useful tool for the assignment of gene function in S. equi and other streptococci. The shared essential gene set of group A, B and C streptococci provides further evidence of the close genetic relationships between these important pathogenic bacteria. Therefore, the ABC of gene essentiality reported here provides a solid foundation towards reporting the functional genome of streptococci.

Library Online Systems.

ERIC Educational Resources Information Center

Folda, Linda; And Others

1989-01-01

Issues related to library online systems are discussed in six articles. Topics covered include staff education through vendor demonstrations, evaluation of online public access catalogs, the impact of integrated online systems on cataloging operations, the merits of smart and dumb barcodes, and points to consider in planning for the next online…

Googling DNA sequences on the World Wide Web.

PubMed

Hajibabaei, Mehrdad; Singer, Gregory A C

2009-11-10

New web-based technologies provide an excellent opportunity for sharing and accessing information and using web as a platform for interaction and collaboration. Although several specialized tools are available for analyzing DNA sequence information, conventional web-based tools have not been utilized for bioinformatics applications. We have developed a novel algorithm and implemented it for searching species-specific genomic sequences, DNA barcodes, by using popular web-based methods such as Google. We developed an alignment independent character based algorithm based on dividing a sequence library (DNA barcodes) and query sequence to words. The actual search is conducted by conventional search tools such as freely available Google Desktop Search. We implemented our algorithm in two exemplar packages. We developed pre and post-processing software to provide customized input and output services, respectively. Our analysis of all publicly available DNA barcode sequences shows a high accuracy as well as rapid results. Our method makes use of conventional web-based technologies for specialized genetic data. It provides a robust and efficient solution for sequence search on the web. The integration of our search method for large-scale sequence libraries such as DNA barcodes provides an excellent web-based tool for accessing this information and linking it to other available categories of information on the web.

PCR cycles above routine numbers do not compromise high-throughput DNA barcoding results.

PubMed

Vierna, J; Doña, J; Vizcaíno, A; Serrano, D; Jovani, R

2017-10-01

High-throughput DNA barcoding has become essential in ecology and evolution, but some technical questions still remain. Increasing the number of PCR cycles above the routine 20-30 cycles is a common practice when working with old-type specimens, which provide little amounts of DNA, or when facing annealing issues with the primers. However, increasing the number of cycles can raise the number of artificial mutations due to polymerase errors. In this work, we sequenced 20 COI libraries in the Illumina MiSeq platform. Libraries were prepared with 40, 45, 50, 55, and 60 PCR cycles from four individuals belonging to four species of four genera of cephalopods. We found no relationship between the number of PCR cycles and the number of mutations despite using a nonproofreading polymerase. Moreover, even when using a high number of PCR cycles, the resulting number of mutations was low enough not to be an issue in the context of high-throughput DNA barcoding (but may still remain an issue in DNA metabarcoding due to chimera formation). We conclude that the common practice of increasing the number of PCR cycles should not negatively impact the outcome of a high-throughput DNA barcoding study in terms of the occurrence of point mutations.

A Barcoding Strategy Enabling Higher-Throughput Library Screening by Microscopy.

PubMed

Chen, Robert; Rishi, Harneet S; Potapov, Vladimir; Yamada, Masaki R; Yeh, Vincent J; Chow, Thomas; Cheung, Celia L; Jones, Austin T; Johnson, Terry D; Keating, Amy E; DeLoache, William C; Dueber, John E

2015-11-20

Dramatic progress has been made in the design and build phases of the design-build-test cycle for engineering cells. However, the test phase usually limits throughput, as many outputs of interest are not amenable to rapid analytical measurements. For example, phenotypes such as motility, morphology, and subcellular localization can be readily measured by microscopy, but analysis of these phenotypes is notoriously slow. To increase throughput, we developed microscopy-readable barcodes (MiCodes) composed of fluorescent proteins targeted to discernible organelles. In this system, a unique barcode can be genetically linked to each library member, making possible the parallel analysis of phenotypes of interest via microscopy. As a first demonstration, we MiCoded a set of synthetic coiled-coil leucine zipper proteins to allow an 8 × 8 matrix to be tested for specific interactions in micrographs consisting of mixed populations of cells. A novel microscopy-readable two-hybrid fluorescence localization assay for probing candidate interactions in the cytosol was also developed using a bait protein targeted to the peroxisome and a prey protein tagged with a fluorescent protein. This work introduces a generalizable, scalable platform for making microscopy amenable to higher-throughput library screening experiments, thereby coupling the power of imaging with the utility of combinatorial search paradigms.

dropEst: pipeline for accurate estimation of molecular counts in droplet-based single-cell RNA-seq experiments.

PubMed

Petukhov, Viktor; Guo, Jimin; Baryawno, Ninib; Severe, Nicolas; Scadden, David T; Samsonova, Maria G; Kharchenko, Peter V

2018-06-19

Recent single-cell RNA-seq protocols based on droplet microfluidics use massively multiplexed barcoding to enable simultaneous measurements of transcriptomes for thousands of individual cells. The increasing complexity of such data creates challenges for subsequent computational processing and troubleshooting of these experiments, with few software options currently available. Here, we describe a flexible pipeline for processing droplet-based transcriptome data that implements barcode corrections, classification of cell quality, and diagnostic information about the droplet libraries. We introduce advanced methods for correcting composition bias and sequencing errors affecting cellular and molecular barcodes to provide more accurate estimates of molecular counts in individual cells.

Spiders (Araneae) of Churchill, Manitoba: DNA barcodes and morphology reveal high species diversity and new Canadian records

PubMed Central

2013-01-01

Background Arctic ecosystems, especially those near transition zones, are expected to be strongly impacted by climate change. Because it is positioned on the ecotone between tundra and boreal forest, the Churchill area is a strategic locality for the analysis of shifts in faunal composition. This fact has motivated the effort to develop a comprehensive biodiversity inventory for the Churchill region by coupling DNA barcoding with morphological studies. The present study represents one element of this effort; it focuses on analysis of the spider fauna at Churchill. Results 198 species were detected among 2704 spiders analyzed, tripling the count for the Churchill region. Estimates of overall diversity suggest that another 10–20 species await detection. Most species displayed little intraspecific sequence variation (maximum <1%) in the barcode region of the cytochrome c oxidase subunit I (COI) gene, but four species showed considerably higher values (maximum = 4.1-6.2%), suggesting cryptic species. All recognized species possessed a distinct haplotype array at COI with nearest-neighbour interspecific distances averaging 8.57%. Three species new to Canada were detected: Robertus lyrifer (Theridiidae), Baryphyma trifrons (Linyphiidae), and Satilatlas monticola (Linyphiidae). The first two species may represent human-mediated introductions linked to the port in Churchill, but the other species represents a range extension from the USA. The first description of the female of S. monticola was also presented. As well, one probable new species of Alopecosa (Lycosidae) was recognized. Conclusions This study provides the first comprehensive DNA barcode reference library for the spider fauna of any region. Few cryptic species of spiders were detected, a result contrasting with the prevalence of undescribed species in several other terrestrial arthropod groups at Churchill. Because most (97.5%) sequence clusters at COI corresponded with a named taxon, DNA barcoding reliably identifies spiders in the Churchill fauna. The capacity of DNA barcoding to enable the identification of otherwise taxonomically ambiguous specimens (juveniles, females) also represents a major advance for future monitoring efforts on this group. PMID:24279427

DNA barcoding detected improper labelling and supersession of crab food served by restaurants in India.

PubMed

Vartak, Vivek Rohidas; Narasimmalu, Rajendran; Annam, Pavan Kumar; Singh, Dhirendra P; Lakra, Wazir S

2015-01-01

Detection of improper labelling of raw and processed seafood is of global importance for reducing commercial fraud and enhancing food safety. Crabs are crustaceans with intricate morphological as well as genetic divergence among species and are popular as seafood in restaurants. Owing to the high number of crab species available, it can be difficult to identify those included in particular food dishes, thus increasing the chance of supersession. DNA barcoding is an advanced technology for detecting improper food labelling and has been used successfully to authenticate seafood. This study identified 11 edible crab species from India by classical taxonomy and developed molecular barcodes with the cytochrome c oxidase I (COI) gene. These barcodes were used as reference barcodes for detecting any improper labelling of 50 restaurant crab samples. Neighbour-joining tree analysis with COI barcodes showed distinct clusters of restaurant samples with respective reference species. The study demonstrated 100% improper labelling of restaurant samples to cover up acts of inferior crab supersession. DNA barcoding successfully identified 11 edible crabs in accordance with classical taxonomy and discerned improper crab food labelling in restaurants of India. © 2014 Society of Chemical Industry.

Digitally encoded DNA nanostructures for multiplexed, single-molecule protein sensing with nanopores

NASA Astrophysics Data System (ADS)

Bell, Nicholas A. W.; Keyser, Ulrich F.

2016-07-01

The simultaneous detection of a large number of different analytes is important in bionanotechnology research and in diagnostic applications. Nanopore sensing is an attractive method in this regard as the approach can be integrated into small, portable device architectures, and there is significant potential for detecting multiple sub-populations in a sample. Here, we show that highly multiplexed sensing of single molecules can be achieved with solid-state nanopores by using digitally encoded DNA nanostructures. Based on the principles of DNA origami, we designed a library of DNA nanostructures in which each member contains a unique barcode; each bit in the barcode is signalled by the presence or absence of multiple DNA dumbbell hairpins. We show that a 3-bit barcode can be assigned with 94% accuracy by electrophoretically driving the DNA structures through a solid-state nanopore. Select members of the library were then functionalized to detect a single, specific antibody through antigen presentation at designed positions on the DNA. This allows us to simultaneously detect four different antibodies of the same isotype at nanomolar concentration levels.

Digitally encoded DNA nanostructures for multiplexed, single-molecule protein sensing with nanopores.

PubMed

Bell, Nicholas A W; Keyser, Ulrich F

2016-07-01

The simultaneous detection of a large number of different analytes is important in bionanotechnology research and in diagnostic applications. Nanopore sensing is an attractive method in this regard as the approach can be integrated into small, portable device architectures, and there is significant potential for detecting multiple sub-populations in a sample. Here, we show that highly multiplexed sensing of single molecules can be achieved with solid-state nanopores by using digitally encoded DNA nanostructures. Based on the principles of DNA origami, we designed a library of DNA nanostructures in which each member contains a unique barcode; each bit in the barcode is signalled by the presence or absence of multiple DNA dumbbell hairpins. We show that a 3-bit barcode can be assigned with 94% accuracy by electrophoretically driving the DNA structures through a solid-state nanopore. Select members of the library were then functionalized to detect a single, specific antibody through antigen presentation at designed positions on the DNA. This allows us to simultaneously detect four different antibodies of the same isotype at nanomolar concentration levels.

DNA barcodes of the native ray-finned fishes in Taiwan.

PubMed

Chang, Chia-Hao; Shao, Kwang-Tsao; Lin, Han-Yang; Chiu, Yung-Chieh; Lee, Mao-Ying; Liu, Shih-Hui; Lin, Pai-Lei

2017-07-01

Species identification based on the DNA sequence of a fragment of the cytochrome c oxidase subunit I gene in the mitochondrial genome, DNA barcoding, is widely applied to assist in sustainable exploitation of fish resources and the protection of fish biodiversity. The aim of this study was to establish a reliable barcoding reference database of the native ray-finned fishes in Taiwan. A total of 2993 individuals, belonging to 1245 species within 637 genera, 184 families and 29 orders of ray-finned fishes and representing approximately 40% of the recorded ray-finned fishes in Taiwan, were PCR amplified at the barcode region and bidirectionally sequenced. The mean length of the 2993 barcodes is 549 bp. Mean congeneric K2P distance (15.24%) is approximately 10-fold higher than the mean conspecific one (1.51%), but approximately 1.4-fold less than the mean genetic distance between families (20.80%). The Barcode Index Number (BIN) discordance report shows that 2993 specimens represent 1275 BINs and, among them, 86 BINs are singletons, 570 BINs are taxonomically concordant, and the other 619 BINs are taxonomically discordant. Barcode gap analysis also revealed that more than 90% of the collected fishes in this study can be discriminated by DNA barcoding. Overall, the barcoding reference database established by this study reveals the need for taxonomic revisions and voucher specimen rechecks, in addition to assisting in the management of Taiwan's fish resources and diversity. © 2016 John Wiley & Sons Ltd.

DNA barcodes for Nearctic Auchenorrhyncha (Insecta: Hemiptera).

PubMed

Foottit, Robert G; Maw, Eric; Hebert, P D N

2014-01-01

Many studies have shown the suitability of sequence variation in the 5' region of the mitochondrial cytochrome c oxidase I (COI) gene as a DNA barcode for the identification of species in a wide range of animal groups. We examined 471 species in 147 genera of Hemiptera: Auchenorrhyncha drawn from specimens in the Canadian National Collection of Insects to assess the effectiveness of DNA barcoding in this group. Analysis of the COI gene revealed less than 2% intra-specific divergence in 93% of the taxa examined, while minimum interspecific distances exceeded 2% in 70% of congeneric species pairs. Although most species are characterized by a distinct sequence cluster, sequences for members of many groups of closely related species either shared sequences or showed close similarity, with 25% of species separated from their nearest neighbor by less than 1%. This study, although preliminary, provides DNA barcodes for about 8% of the species of this hemipteran suborder found in North America north of Mexico. Barcodes can enable the identification of many species of Auchenorrhyncha, but members of some species groups cannot be discriminated. Future use of DNA barcodes in regulatory, pest management, and environmental applications will be possible as the barcode library for Auchenorrhyncha expands to include more species and broader geographic coverage.

DNA Barcodes for Nearctic Auchenorrhyncha (Insecta: Hemiptera)

PubMed Central

Foottit, Robert G.; Maw, Eric; Hebert, P. D. N.

2014-01-01

Background Many studies have shown the suitability of sequence variation in the 5′ region of the mitochondrial cytochrome c oxidase I (COI) gene as a DNA barcode for the identification of species in a wide range of animal groups. We examined 471 species in 147 genera of Hemiptera: Auchenorrhyncha drawn from specimens in the Canadian National Collection of Insects to assess the effectiveness of DNA barcoding in this group. Methodology/Principal Findings Analysis of the COI gene revealed less than 2% intra-specific divergence in 93% of the taxa examined, while minimum interspecific distances exceeded 2% in 70% of congeneric species pairs. Although most species are characterized by a distinct sequence cluster, sequences for members of many groups of closely related species either shared sequences or showed close similarity, with 25% of species separated from their nearest neighbor by less than 1%. Conclusions/Significance This study, although preliminary, provides DNA barcodes for about 8% of the species of this hemipteran suborder found in North America north of Mexico. Barcodes can enable the identification of many species of Auchenorrhyncha, but members of some species groups cannot be discriminated. Future use of DNA barcodes in regulatory, pest management, and environmental applications will be possible as the barcode library for Auchenorrhyncha expands to include more species and broader geographic coverage. PMID:25004106

High-throughput, image-based screening of pooled genetic variant libraries

PubMed Central

Emanuel, George; Moffitt, Jeffrey R.; Zhuang, Xiaowei

2018-01-01

Image-based, high-throughput screening of genetic perturbations will advance both biology and biotechnology. We report a high-throughput screening method that allows diverse genotypes and corresponding phenotypes to be imaged in numerous individual cells. We achieve genotyping by introducing barcoded genetic variants into cells and using massively multiplexed FISH to measure the barcodes. We demonstrated this method by screening mutants of the fluorescent protein YFAST, yielding brighter and more photostable YFAST variants. PMID:29083401

Towards monitoring the sandflies (Diptera: Psychodidae) of Thailand: DNA barcoding the sandflies of Wihan Cave, Uttaradit.

PubMed

Polseela, Raxsina; Jaturas, Narong; Thanwisai, Aunchalee; Sing, Kong-Wah; Wilson, John-James

2016-09-01

Sandflies vary in their distributions and role in pathogen transmission. Attempts to record distributions of sandflies in Thailand have faced difficulties due to their high abundance and diversity. We aim to provide an insight into the diversity of sandflies in Thailand by (i) conducting a literature review, and (ii) DNA barcoding sandflies collected from Wihan Cave where eight morphologically characterized species were recorded. DNA barcodes generated for 193 sandflies fell into 13 distinct species clusters under four genera (Chinius, Idiophlebotomus, Phlebotomus and Sergentomyia). Five of these species could be assigned Linnaean species names unambiguously and two others corresponded to characterized morphospecies. Two species represented a complex under the name Sergentomyia barraudi while the remaining four had not been recognized before in any form. The resulting species checklist and DNA barcode library contribute to a growing set of records for sandflies which is useful for monitoring and vector control.

Utility of DNA barcoding for rapid and accurate assessment of bat diversity in Malaysia in the absence of formally described species.

PubMed

Wilson, J-J; Sing, K-W; Halim, M R A; Ramli, R; Hashim, R; Sofian-Azirun, M

2014-02-19

Bats are important flagship species for biodiversity research; however, diversity in Southeast Asia is considerably underestimated in the current checklists and field guides. Incorporation of DNA barcoding into surveys has revealed numerous species-level taxa overlooked by conventional methods. Inclusion of these taxa in inventories provides a more informative record of diversity, but is problematic as these species lack formal description. We investigated how frequently documented, but undescribed, bat taxa are encountered in Peninsular Malaysia. We discuss whether a barcode library provides a means of recognizing and recording these taxa across biodiversity inventories. Tissue was sampled from bats trapped at Pasir Raja, Dungun Terengganu, Peninsular Malaysia. The DNA was extracted and the COI barcode region amplified and sequenced. We identified 9 species-level taxa within our samples, based on analysis of the DNA barcodes. Six specimens matched to four previously documented taxa considered candidate species but currently lacking formal taxonomic status. This study confirms the high diversity of bats within Peninsular Malaysia (9 species in 13 samples) and demonstrates how DNA barcoding allows for inventory and documentation of known taxa lacking formal taxonomic status.

Delineating Species with DNA Barcodes: A Case of Taxon Dependent Method Performance in Moths

PubMed Central

Kekkonen, Mari; Mutanen, Marko; Kaila, Lauri; Nieminen, Marko; Hebert, Paul D. N.

2015-01-01

The accelerating loss of biodiversity has created a need for more effective ways to discover species. Novel algorithmic approaches for analyzing sequence data combined with rapidly expanding DNA barcode libraries provide a potential solution. While several analytical methods are available for the delineation of operational taxonomic units (OTUs), few studies have compared their performance. This study compares the performance of one morphology-based and four DNA-based (BIN, parsimony networks, ABGD, GMYC) methods on two groups of gelechioid moths. It examines 92 species of Finnish Gelechiinae and 103 species of Australian Elachistinae which were delineated by traditional taxonomy. The results reveal a striking difference in performance between the two taxa with all four DNA-based methods. OTU counts in the Elachistinae showed a wider range and a relatively low (ca. 65%) OTU match with reference species while OTU counts were more congruent and performance was higher (ca. 90%) in the Gelechiinae. Performance rose when only monophyletic species were compared, but the taxon-dependence remained. None of the DNA-based methods produced a correct match with non-monophyletic species, but singletons were handled well. A simulated test of morphospecies-grouping performed very poorly in revealing taxon diversity in these small, dull-colored moths. Despite the strong performance of analyses based on DNA barcodes, species delineated using single-locus mtDNA data are best viewed as OTUs that require validation by subsequent integrative taxonomic work. PMID:25849083

Ultra-deep sequencing enables high-fidelity recovery of biodiversity for bulk arthropod samples without PCR amplification

PubMed Central

2013-01-01

Background Next-generation-sequencing (NGS) technologies combined with a classic DNA barcoding approach have enabled fast and credible measurement for biodiversity of mixed environmental samples. However, the PCR amplification involved in nearly all existing NGS protocols inevitably introduces taxonomic biases. In the present study, we developed new Illumina pipelines without PCR amplifications to analyze terrestrial arthropod communities. Results Mitochondrial enrichment directly followed by Illumina shotgun sequencing, at an ultra-high sequence volume, enabled the recovery of Cytochrome c Oxidase subunit 1 (COI) barcode sequences, which allowed for the estimation of species composition at high fidelity for a terrestrial insect community. With 15.5 Gbp Illumina data, approximately 97% and 92% were detected out of the 37 input Operational Taxonomic Units (OTUs), whether the reference barcode library was used or not, respectively, while only 1 novel OTU was found for the latter. Additionally, relatively strong correlation between the sequencing volume and the total biomass was observed for species from the bulk sample, suggesting a potential solution to reveal relative abundance. Conclusions The ability of the new Illumina PCR-free pipeline for DNA metabarcoding to detect small arthropod specimens and its tendency to avoid most, if not all, false positives suggests its great potential in biodiversity-related surveillance, such as in biomonitoring programs. However, further improvement for mitochondrial enrichment is likely needed for the application of the new pipeline in analyzing arthropod communities at higher diversity. PMID:23587339

Scalable Amplification of Strand Subsets from Chip-Synthesized Oligonucleotide Libraries (Open Access)

DTIC Science & Technology

2015-11-16

detailed discussion of barcode designs in Supplementary Note 1, Supplementary Fig. 1 and sequences in Supplementary Note 2). Whereas the nicking and...eight subpools, each as a one- or as a two-barcode version ( design details in Supplementary Note 1). All subpools amplified strands with the expected...for the c2ca designs . We used the same restriction enzymes (Nb.BsrDI and Nt.BspQI) that were encoded between the primers and the target sequences to

The Microbial Ferrous Wheel in a Neutral pH Groundwater Seep

PubMed Central

Roden, Eric E.; McBeth, Joyce M.; Blöthe, Marco; Percak-Dennett, Elizabeth M.; Fleming, Emily J.; Holyoke, Rebecca R.; Luther, George W.; Emerson, David; Schieber, Juergen

2012-01-01

Evidence for microbial Fe redox cycling was documented in a circumneutral pH groundwater seep near Bloomington, Indiana. Geochemical and microbiological analyses were conducted at two sites, a semi-consolidated microbial mat and a floating puffball structure. In situ voltammetric microelectrode measurements revealed steep opposing gradients of O2 and Fe(II) at both sites, similar to other groundwater seep and sedimentary environments known to support microbial Fe redox cycling. The puffball structure showed an abrupt increase in dissolved Fe(II) just at its surface (∼5 cm depth), suggesting an internal Fe(II) source coupled to active Fe(III) reduction. Most probable number enumerations detected microaerophilic Fe(II)-oxidizing bacteria (FeOB) and dissimilatory Fe(III)-reducing bacteria (FeRB) at densities of 102 to 105 cells mL−1 in samples from both sites. In vitro Fe(III) reduction experiments revealed the potential for immediate reduction (no lag period) of native Fe(III) oxides. Conventional full-length 16S rRNA gene clone libraries were compared with high throughput barcode sequencing of the V1, V4, or V6 variable regions of 16S rRNA genes in order to evaluate the extent to which new sequencing approaches could provide enhanced insight into the composition of Fe redox cycling microbial community structure. The composition of the clone libraries suggested a lithotroph-dominated microbial community centered around taxa related to known FeOB (e.g., Gallionella, Sideroxydans, Aquabacterium). Sequences related to recognized FeRB (e.g., Rhodoferax, Aeromonas, Geobacter, Desulfovibrio) were also well-represented. Overall, sequences related to known FeOB and FeRB accounted for 88 and 59% of total clone sequences in the mat and puffball libraries, respectively. Taxa identified in the barcode libraries showed partial overlap with the clone libraries, but were not always consistent across different variable regions and sequencing platforms. However, the barcode libraries provided confirmation of key clone library results (e.g., the predominance of Betaproteobacteria) and an expanded view of lithotrophic microbial community composition. PMID:22783228

Barcoding Fauna Bavarica: 78% of the Neuropterida Fauna Barcoded!

PubMed Central

Morinière, Jérome; Hendrich, Lars; Hausmann, Axel; Hebert, Paul; Haszprunar, Gerhard; Gruppe, Axel

2014-01-01

This publication provides the first comprehensive DNA barcode data set for the Neuropterida of Central Europe, including 80 of the 102 species (78%) recorded from Bavaria (Germany) and three other species from nearby regions (Austria, France and the UK). Although the 286 specimens analyzed had a heterogeneous conservation history (60% dried; 30% in 80% EtOH; 10% fresh specimens in 95% EtOH), 237 (83%) generated a DNA barcode. Eleven species (13%) shared a BIN, but three of these taxa could be discriminated through barcodes. Four pairs of closely allied species shared barcodes including Chrysoperla pallida Henry et al., 2002 and C. lucasina Lacroix, 1912; Wesmaelius concinnus (Stephens, 1836) and W. quadrifasciatus (Reuter, 1894); Hemerobius handschini Tjeder, 1957 and H. nitidulus Fabricius, 1777; and H. atrifrons McLachlan, 1868 and H. contumax Tjeder, 1932. Further studies are needed to test the possible synonymy of these species pairs or to determine if other genetic markers permit their discrimination. Our data highlight five cases of potential cryptic diversity within Bavarian Neuropterida: Nineta flava (Scopoli, 1763), Sympherobius pygmaeus (Rambur, 1842), Sisyra nigra (Retzius, 1783), Semidalis aleyrodiformis (Stephens, 1836) and Coniopteryx pygmaea Enderlein, 1906 are each split into two or three BINs. The present DNA barcode library not only allows the identification of adult and larval stages, but also provides valuable information for alpha-taxonomy, and for ecological and evolutionary research. PMID:25286434

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens.

PubMed

Loudig, Olivier; Liu, Christina; Rohan, Thomas; Ben-Dov, Iddo Z

2018-05-05

-Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of cancer development. By using non-invasive or pre-malignant lesions from patients who later develop invasive disease, gene expression analyses may help identify early molecular alterations that predispose to cancer risk. It has been well described that nucleic acids recovered from FFPE tissues have undergone severe physical damage and chemical modifications, which make their analysis difficult and generally requires adapted assays. MicroRNAs (miRNAs), however, which represent a small class of RNA molecules spanning only up to ~18-24 nucleotides, have been shown to withstand long-term storage and have been successfully analyzed in FFPE samples. Here we present a 3' barcoded complementary DNA (cDNA) library preparation protocol specifically optimized for the analysis of small RNAs extracted from archived tissues, which was recently demonstrated to be robust and highly reproducible when using archived clinical specimens stored for up to 35 years. This library preparation is well adapted to the multiplex analysis of compromised/degraded material where RNA samples (up to 18) are ligated with individual 3' barcoded adapters and then pooled together for subsequent enzymatic and biochemical preparations prior to analysis. All purifications are performed by polyacrylamide gel electrophoresis (PAGE), which allows size-specific selections and enrichments of barcoded small RNA species. This cDNA library preparation is well adapted to minute RNA inputs, as a pilot polymerase chain reaction (PCR) allows determination of a specific amplification cycle to produce optimal amounts of material for next-generation sequencing (NGS). This approach was optimized for the use of degraded FFPE RNA from specimens archived for up to 35 years and provides highly reproducible NGS data.

A Universal Next-Generation Sequencing Protocol To Generate Noninfectious Barcoded cDNA Libraries from High-Containment RNA Viruses

PubMed Central

Moser, Lindsey A.; Ramirez-Carvajal, Lisbeth; Puri, Vinita; Pauszek, Steven J.; Matthews, Krystal; Dilley, Kari A.; Mullan, Clancy; McGraw, Jennifer; Khayat, Michael; Beeri, Karen; Yee, Anthony; Dugan, Vivien; Heise, Mark T.; Frieman, Matthew B.; Rodriguez, Luis L.; Bernard, Kristen A.; Wentworth, David E.

2016-01-01

ABSTRACT Several biosafety level 3 and/or 4 (BSL-3/4) pathogens are high-consequence, single-stranded RNA viruses, and their genomes, when introduced into permissive cells, are infectious. Moreover, many of these viruses are select agents (SAs), and their genomes are also considered SAs. For this reason, cDNAs and/or their derivatives must be tested to ensure the absence of infectious virus and/or viral RNA before transfer out of the BSL-3/4 and/or SA laboratory. This tremendously limits the capacity to conduct viral genomic research, particularly the application of next-generation sequencing (NGS). Here, we present a sequence-independent method to rapidly amplify viral genomic RNA while simultaneously abolishing both viral and genomic RNA infectivity across multiple single-stranded positive-sense RNA (ssRNA+) virus families. The process generates barcoded DNA amplicons that range in length from 300 to 1,000 bp, which cannot be used to rescue a virus and are stable to transport at room temperature. Our barcoding approach allows for up to 288 barcoded samples to be pooled into a single library and run across various NGS platforms without potential reconstitution of the viral genome. Our data demonstrate that this approach provides full-length genomic sequence information not only from high-titer virion preparations but it can also recover specific viral sequence from samples with limited starting material in the background of cellular RNA, and it can be used to identify pathogens from unknown samples. In summary, we describe a rapid, universal standard operating procedure that generates high-quality NGS libraries free of infectious virus and infectious viral RNA. IMPORTANCE This report establishes and validates a standard operating procedure (SOP) for select agents (SAs) and other biosafety level 3 and/or 4 (BSL-3/4) RNA viruses to rapidly generate noninfectious, barcoded cDNA amenable for next-generation sequencing (NGS). This eliminates the burden of testing all processed samples derived from high-consequence pathogens prior to transfer from high-containment laboratories to lower-containment facilities for sequencing. Our established protocol can be scaled up for high-throughput sequencing of hundreds of samples simultaneously, which can dramatically reduce the cost and effort required for NGS library construction. NGS data from this SOP can provide complete genome coverage from viral stocks and can also detect virus-specific reads from limited starting material. Our data suggest that the procedure can be implemented and easily validated by institutional biosafety committees across research laboratories. PMID:27822536

Defining operational taxonomic units using DNA barcode data.

PubMed

Blaxter, Mark; Mann, Jenna; Chapman, Tom; Thomas, Fran; Whitton, Claire; Floyd, Robin; Abebe, Eyualem

2005-10-29

The scale of diversity of life on this planet is a significant challenge for any scientific programme hoping to produce a complete catalogue, whatever means is used. For DNA barcoding studies, this difficulty is compounded by the realization that any chosen barcode sequence is not the gene 'for' speciation and that taxa have evolutionary histories. How are we to disentangle the confounding effects of reticulate population genetic processes? Using the DNA barcode data from meiofaunal surveys, here we discuss the benefits of treating the taxa defined by barcodes without reference to their correspondence to 'species', and suggest that using this non-idealist approach facilitates access to taxon groups that are not accessible to other methods of enumeration and classification. Major issues remain, in particular the methodologies for taxon discrimination in DNA barcode data.

Testing efficacy of distance and tree-based methods for DNA barcoding of grasses (Poaceae tribe Poeae) in Australia

PubMed Central

Walsh, Neville G.; Cantrill, David J.; Holmes, Gareth D.; Murphy, Daniel J.

2017-01-01

In Australia, Poaceae tribe Poeae are represented by 19 genera and 99 species, including economically and environmentally important native and introduced pasture grasses [e.g. Poa (Tussock-grasses) and Lolium (Ryegrasses)]. We used this tribe, which are well characterised in regards to morphological diversity and evolutionary relationships, to test the efficacy of DNA barcoding methods. A reference library was generated that included 93.9% of species in Australia (408 individuals, x¯ = 3.7 individuals per species). Molecular data were generated for official plant barcoding markers (rbcL, matK) and the nuclear ribosomal internal transcribed spacer (ITS) region. We investigated accuracy of specimen identifications using distance- (nearest neighbour, best-close match, and threshold identification) and tree-based (maximum likelihood, Bayesian inference) methods and applied species discovery methods (automatic barcode gap discovery, Poisson tree processes) based on molecular data to assess congruence with recognised species. Across all methods, success rate for specimen identification of genera was high (87.5–99.5%) and of species was low (25.6–44.6%). Distance- and tree-based methods were equally ineffective in providing accurate identifications for specimens to species rank (26.1–44.6% and 25.6–31.3%, respectively). The ITS marker achieved the highest success rate for specimen identification at both generic and species ranks across the majority of methods. For distance-based analyses the best-close match method provided the greatest accuracy for identification of individuals with a high percentage of “correct” (97.6%) and a low percentage of “incorrect” (0.3%) generic identifications, based on the ITS marker. For tribe Poeae, and likely for other grass lineages, sequence data in the standard DNA barcode markers are not variable enough for accurate identification of specimens to species rank. For recently diverged grass species similar challenges are encountered in the application of genetic and morphological data to species delimitations, with taxonomic signal limited by extensive infra-specific variation and shared polymorphisms among species in both data types. PMID:29084279

DNA barcoding for species identification from dried and powdered plant parts: a case study with authentication of the raw drug market samples of Sida cordifolia.

PubMed

Vassou, Sophie Lorraine; Kusuma, G; Parani, Madasamy

2015-03-15

The majority of the plant materials used in herbal medicine is procured from the markets in the form of dried or powdered plant parts. It is essential to use authentic plant materials to derive the benefits of herbal medicine. However, establishing the identity of these plant materials by conventional taxonomy is extremely difficult. Here we report a case study in which the species identification of the market samples of Sida cordifolia was done by DNA barcoding. As a prelude to species identification by DNA barcoding, 13 species of Sida were collected, and a reference DNA barcode library was developed using rbcL, matK, psbA-trnH and ITS2 markers. Based on the intra-species and inter-species divergence observed, psbA-trnH and ITS2 were found to be the best two-marker combination for species identification of the market samples. The study showed that none of the market samples belonged to the authentic species, S. cordifolia. Seventy-six per cent of the market samples belonged to other species of Sida. The predominant one was Sida acuta (36%) followed by S. spinosa (20%), S. alnifolia (12%), S. scabrida (4%) and S. ravii (4%). Such substitutions may not only fail to give the expected therapeutic effect, but may also give undesirable effects as in case of S. acuta which contains a 6-fold higher amount of ephedrine compared to the roots of S. cordifolia. The remaining 24% of the samples were from other genera such as Abutilon sp. (8%), Ixonanthes sp., Terminalia sp., Fagonia sp., and Tephrosia sp. (4% each). This observation is in contrast to the belief that medicinal plants are generally substituted or adulterated with closely related species. The current study strongly suggests that the raw drug market samples of herbal medicines need to be properly authenticated before use, and DNA barcoding has been found to be suitable for this purpose. Copyright © 2015 Elsevier B.V. All rights reserved.

Testing efficacy of distance and tree-based methods for DNA barcoding of grasses (Poaceae tribe Poeae) in Australia.

PubMed

Birch, Joanne L; Walsh, Neville G; Cantrill, David J; Holmes, Gareth D; Murphy, Daniel J

2017-01-01

In Australia, Poaceae tribe Poeae are represented by 19 genera and 99 species, including economically and environmentally important native and introduced pasture grasses [e.g. Poa (Tussock-grasses) and Lolium (Ryegrasses)]. We used this tribe, which are well characterised in regards to morphological diversity and evolutionary relationships, to test the efficacy of DNA barcoding methods. A reference library was generated that included 93.9% of species in Australia (408 individuals, [Formula: see text] = 3.7 individuals per species). Molecular data were generated for official plant barcoding markers (rbcL, matK) and the nuclear ribosomal internal transcribed spacer (ITS) region. We investigated accuracy of specimen identifications using distance- (nearest neighbour, best-close match, and threshold identification) and tree-based (maximum likelihood, Bayesian inference) methods and applied species discovery methods (automatic barcode gap discovery, Poisson tree processes) based on molecular data to assess congruence with recognised species. Across all methods, success rate for specimen identification of genera was high (87.5-99.5%) and of species was low (25.6-44.6%). Distance- and tree-based methods were equally ineffective in providing accurate identifications for specimens to species rank (26.1-44.6% and 25.6-31.3%, respectively). The ITS marker achieved the highest success rate for specimen identification at both generic and species ranks across the majority of methods. For distance-based analyses the best-close match method provided the greatest accuracy for identification of individuals with a high percentage of "correct" (97.6%) and a low percentage of "incorrect" (0.3%) generic identifications, based on the ITS marker. For tribe Poeae, and likely for other grass lineages, sequence data in the standard DNA barcode markers are not variable enough for accurate identification of specimens to species rank. For recently diverged grass species similar challenges are encountered in the application of genetic and morphological data to species delimitations, with taxonomic signal limited by extensive infra-specific variation and shared polymorphisms among species in both data types.

Enhancing the detection of barcoded reads in high throughput DNA sequencing data by controlling the false discovery rate.

PubMed

Buschmann, Tilo; Zhang, Rong; Brash, Douglas E; Bystrykh, Leonid V

2014-08-07

DNA barcodes are short unique sequences used to label DNA or RNA-derived samples in multiplexed deep sequencing experiments. During the demultiplexing step, barcodes must be detected and their position identified. In some cases (e.g., with PacBio SMRT), the position of the barcode and DNA context is not well defined. Many reads start inside the genomic insert so that adjacent primers might be missed. The matter is further complicated by coincidental similarities between barcode sequences and reference DNA. Therefore, a robust strategy is required in order to detect barcoded reads and avoid a large number of false positives or negatives.For mass inference problems such as this one, false discovery rate (FDR) methods are powerful and balanced solutions. Since existing FDR methods cannot be applied to this particular problem, we present an adapted FDR method that is suitable for the detection of barcoded reads as well as suggest possible improvements. In our analysis, barcode sequences showed high rates of coincidental similarities with the Mus musculus reference DNA. This problem became more acute when the length of the barcode sequence decreased and the number of barcodes in the set increased. The method presented in this paper controls the tail area-based false discovery rate to distinguish between barcoded and unbarcoded reads. This method helps to establish the highest acceptable minimal distance between reads and barcode sequences. In a proof of concept experiment we correctly detected barcodes in 83% of the reads with a precision of 89%. Sensitivity improved to 99% at 99% precision when the adjacent primer sequence was incorporated in the analysis. The analysis was further improved using a paired end strategy. Following an analysis of the data for sequence variants induced in the Atp1a1 gene of C57BL/6 murine melanocytes by ultraviolet light and conferring resistance to ouabain, we found no evidence of cross-contamination of DNA material between samples. Our method offers a proper quantitative treatment of the problem of detecting barcoded reads in a noisy sequencing environment. It is based on the false discovery rate statistics that allows a proper trade-off between sensitivity and precision to be chosen.

DNA barcodes for two scale insect families, mealybugs (Hemiptera: Pseudococcidae) and armored scales (Hemiptera: Diaspididae).

PubMed

Park, D-S; Suh, S-J; Hebert, P D N; Oh, H-W; Hong, K-J

2011-08-01

Although DNA barcode coverage has grown rapidly for many insect orders, there are some groups, such as scale insects, where sequence recovery has been difficult. However, using a recently developed primer set, we recovered barcode records from 373 specimens, providing coverage for 75 species from 31 genera in two families. Overall success was >90% for mealybugs and >80% for armored scale species. The G·C content was very low in most species, averaging just 16.3%. Sequence divergences (K2P) between congeneric species averaged 10.7%, while intra-specific divergences averaged 0.97%. However, the latter value was inflated by high intra-specific divergence in nine taxa, cases that may indicate species overlooked by current taxonomic treatments. Our study establishes the feasibility of developing a comprehensive barcode library for scale insects and indicates that its construction will both create an effective system for identifying scale insects and reveal taxonomic situations worthy of deeper analysis.

Spatial heterogeneity in the Mediterranean Biodiversity Hotspot affects barcoding accuracy of its freshwater fishes.

PubMed

Geiger, M F; Herder, F; Monaghan, M T; Almada, V; Barbieri, R; Bariche, M; Berrebi, P; Bohlen, J; Casal-Lopez, M; Delmastro, G B; Denys, G P J; Dettai, A; Doadrio, I; Kalogianni, E; Kärst, H; Kottelat, M; Kovačić, M; Laporte, M; Lorenzoni, M; Marčić, Z; Özuluğ, M; Perdices, A; Perea, S; Persat, H; Porcelotti, S; Puzzi, C; Robalo, J; Šanda, R; Schneider, M; Šlechtová, V; Stoumboudi, M; Walter, S; Freyhof, J

2014-11-01

Incomplete knowledge of biodiversity remains a stumbling block for conservation planning and even occurs within globally important Biodiversity Hotspots (BH). Although technical advances have boosted the power of molecular biodiversity assessments, the link between DNA sequences and species and the analytics to discriminate entities remain crucial. Here, we present an analysis of the first DNA barcode library for the freshwater fish fauna of the Mediterranean BH (526 spp.), with virtually complete species coverage (498 spp., 98% extant species). In order to build an identification system supporting conservation, we compared species determination by taxonomists to multiple clustering analyses of DNA barcodes for 3165 specimens. The congruence of barcode clusters with morphological determination was strongly dependent on the method of cluster delineation, but was highest with the general mixed Yule-coalescent (GMYC) model-based approach (83% of all species recovered as GMYC entity). Overall, genetic morphological discontinuities suggest the existence of up to 64 previously unrecognized candidate species. We found reduced identification accuracy when using the entire DNA-barcode database, compared with analyses on databases for individual river catchments. This scale effect has important implications for barcoding assessments and suggests that fairly simple identification pipelines provide sufficient resolution in local applications. We calculated Evolutionarily Distinct and Globally Endangered scores in order to identify candidate species for conservation priority and argue that the evolutionary content of barcode data can be used to detect priority species for future IUCN assessments. We show that large-scale barcoding inventories of complex biotas are feasible and contribute directly to the evaluation of conservation priorities. © 2014 John Wiley & Sons Ltd.

Competitive Genomic Screens of Barcoded Yeast Libraries

PubMed Central

Urbanus, Malene; Proctor, Michael; Heisler, Lawrence E.; Giaever, Guri; Nislow, Corey

2011-01-01

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment. PMID:21860376

DNA barcoding as a useful tool in the systematic study of wild bees of the tribe Augochlorini (Hymenoptera: Halictidae).

PubMed

González-Vaquero, Rocío Ana; Roig-Alsina, Arturo; Packer, Laurence

2016-10-01

Special care is needed in the delimitation and identification of halictid bee species, which are renowned for being morphologically monotonous. Corynura Spinola and Halictillus Moure (Halictidae: Augochlorini) contain species that are key elements in southern South American ecosystems. These bees are very difficult to identify due to close morphological similarity among species and high sexual dimorphism. We analyzed 170 barcode-compliant COI sequences from 19 species. DNA barcodes were useful to confirm gender associations and to detect two new cryptic species. Interspecific distances were significantly higher than those reported for other bees. Maximum intraspecific divergence was less than 1% in 14 species. Barcode index numbers (BINs) were useful to identify putative species that need further study. More than one BIN was assigned to five species. The name Corynura patagonica (Cockerell) probably refers to two cryptic species. The results suggest that Corynura and Halictillus species can be identified using DNA barcodes. The sequences of the species included in this study can be used as a reference to assess the identification of unknown specimens. This study provides additional support for the use of DNA barcodes in bee taxonomy and the identification of specimens, which is particularly relevant in insects of ecological importance such as pollinators.

Defining operational taxonomic units using DNA barcode data

PubMed Central

Blaxter, Mark; Mann, Jenna; Chapman, Tom; Thomas, Fran; Whitton, Claire; Floyd, Robin; Abebe, Eyualem

2005-01-01

Abstract The scale of diversity of life on this planet is a significant challenge for any scientific programme hoping to produce a complete catalogue, whatever means is used. For DNA barcoding studies, this difficulty is compounded by the realization that any chosen barcode sequence is not the gene ‘for’ speciation and that taxa have evolutionary histories. How are we to disentangle the confounding effects of reticulate population genetic processes? Using the DNA barcode data from meiofaunal surveys, here we discuss the benefits of treating the taxa defined by barcodes without reference to their correspondence to ‘species’, and suggest that using this non-idealist approach facilitates access to taxon groups that are not accessible to other methods of enumeration and classification. Major issues remain, in particular the methodologies for taxon discrimination in DNA barcode data. PMID:16214751

Multiplex single-molecule interaction profiling of DNA barcoded proteins

PubMed Central

Gu, Liangcai; Li, Chao; Aach, John; Hill, David E.; Vidal, Marc; Church, George M.

2014-01-01

In contrast with advances in massively parallel DNA sequencing1, high-throughput protein analyses2-4 are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule (SM) protein detection achieved using optical methods5 is limited by the number of spectrally nonoverlapping chromophores. Here, we introduce a single molecular interaction-sequencing (SMI-Seq) technology for parallel protein interaction profiling leveraging SM advantages. DNA barcodes are attached to proteins collectively via ribosome display6 or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide (PAA) thin film to construct a random SM array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies)7 and analyzed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimeter. Furthermore, protein interactions can be measured based on the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor (GPCR) and antibody binding profiling, were demonstrated. SMI-Seq enables “library vs. library” screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity. PMID:25252978

Mapping Biodiversity and Setting Conservation Priorities for SE Queensland’s Rainforests Using DNA Barcoding

PubMed Central

Shapcott, Alison; Forster, Paul I.; Guymer, Gordon P.; McDonald, William J. F.; Faith, Daniel P.; Erickson, David; Kress, W. John

2015-01-01

Australian rainforests have been fragmented due to past climatic changes and more recently landscape change as a result of clearing for agriculture and urban spread. The subtropical rainforests of South Eastern Queensland are significantly more fragmented than the tropical World Heritage listed northern rainforests and are subject to much greater human population pressures. The Australian rainforest flora is relatively taxonomically rich at the family level, but less so at the species level. Current methods to assess biodiversity based on species numbers fail to adequately capture this richness at higher taxonomic levels. We developed a DNA barcode library for the SE Queensland rainforest flora to support a methodology for biodiversity assessment that incorporates both taxonomic diversity and phylogenetic relationships. We placed our SE Queensland phylogeny based on a three marker DNA barcode within a larger international rainforest barcode library and used this to calculate phylogenetic diversity (PD). We compared phylo- diversity measures, species composition and richness and ecosystem diversity of the SE Queensland rainforest estate to identify which bio subregions contain the greatest rainforest biodiversity, subregion relationships and their level of protection. We identified areas of highest conservation priority. Diversity was not correlated with rainforest area in SE Queensland subregions but PD was correlated with both the percent of the subregion occupied by rainforest and the diversity of regional ecosystems (RE) present. The patterns of species diversity and phylogenetic diversity suggest a strong influence of historical biogeography. Some subregions contain significantly more PD than expected by chance, consistent with the concept of refugia, while others were significantly phylogenetically clustered, consistent with recent range expansions. PMID:25803607

Mapping biodiversity and setting conservation priorities for SE Queensland's rainforests using DNA barcoding.

PubMed

Shapcott, Alison; Forster, Paul I; Guymer, Gordon P; McDonald, William J F; Faith, Daniel P; Erickson, David; Kress, W John

2015-01-01

Australian rainforests have been fragmented due to past climatic changes and more recently landscape change as a result of clearing for agriculture and urban spread. The subtropical rainforests of South Eastern Queensland are significantly more fragmented than the tropical World Heritage listed northern rainforests and are subject to much greater human population pressures. The Australian rainforest flora is relatively taxonomically rich at the family level, but less so at the species level. Current methods to assess biodiversity based on species numbers fail to adequately capture this richness at higher taxonomic levels. We developed a DNA barcode library for the SE Queensland rainforest flora to support a methodology for biodiversity assessment that incorporates both taxonomic diversity and phylogenetic relationships. We placed our SE Queensland phylogeny based on a three marker DNA barcode within a larger international rainforest barcode library and used this to calculate phylogenetic diversity (PD). We compared phylo- diversity measures, species composition and richness and ecosystem diversity of the SE Queensland rainforest estate to identify which bio subregions contain the greatest rainforest biodiversity, subregion relationships and their level of protection. We identified areas of highest conservation priority. Diversity was not correlated with rainforest area in SE Queensland subregions but PD was correlated with both the percent of the subregion occupied by rainforest and the diversity of regional ecosystems (RE) present. The patterns of species diversity and phylogenetic diversity suggest a strong influence of historical biogeography. Some subregions contain significantly more PD than expected by chance, consistent with the concept of refugia, while others were significantly phylogenetically clustered, consistent with recent range expansions.

Single-cell barcoding and sequencing using droplet microfluidics.

PubMed

Zilionis, Rapolas; Nainys, Juozas; Veres, Adrian; Savova, Virginia; Zemmour, David; Klein, Allon M; Mazutis, Linas

2017-01-01

Single-cell RNA sequencing has recently emerged as a powerful tool for mapping cellular heterogeneity in diseased and healthy tissues, yet high-throughput methods are needed for capturing the unbiased diversity of cells. Droplet microfluidics is among the most promising candidates for capturing and processing thousands of individual cells for whole-transcriptome or genomic analysis in a massively parallel manner with minimal reagent use. We recently established a method called inDrops, which has the capability to index >15,000 cells in an hour. A suspension of cells is first encapsulated into nanoliter droplets with hydrogel beads (HBs) bearing barcoding DNA primers. Cells are then lysed and mRNA is barcoded (indexed) by a reverse transcription (RT) reaction. Here we provide details for (i) establishing an inDrops platform (1 d); (ii) performing hydrogel bead synthesis (4 d); (iii) encapsulating and barcoding cells (1 d); and (iv) RNA-seq library preparation (2 d). inDrops is a robust and scalable platform, and it is unique in its ability to capture and profile >75% of cells in even very small samples, on a scale of thousands or tens of thousands of cells.

DNA barcode reference library for Iberian butterflies enables a continental-scale preview of potential cryptic diversity

PubMed Central

Dincă, Vlad; Montagud, Sergio; Talavera, Gerard; Hernández-Roldán, Juan; Munguira, Miguel L.; García-Barros, Enrique; Hebert, Paul D. N.; Vila, Roger

2015-01-01

How common are cryptic species - those overlooked because of their morphological similarity? Despite its wide-ranging implications for biology and conservation, the answer remains open to debate. Butterflies constitute the best-studied invertebrates, playing a similar role as birds do in providing models for vertebrate biology. An accurate assessment of cryptic diversity in this emblematic group requires meticulous case-by-case assessments, but a preview to highlight cases of particular interest will help to direct future studies. We present a survey of mitochondrial genetic diversity for the butterfly fauna of the Iberian Peninsula with unprecedented resolution (3502 DNA barcodes for all 228 species), creating a reliable system for DNA-based identification and for the detection of overlooked diversity. After compiling available data for European butterflies (5782 sequences, 299 species), we applied the Generalized Mixed Yule-Coalescent model to explore potential cryptic diversity at a continental scale. The results indicate that 27.7% of these species include from two to four evolutionary significant units (ESUs), suggesting that cryptic biodiversity may be higher than expected for one of the best-studied invertebrate groups and regions. The ESUs represent important units for conservation, models for studies of evolutionary and speciation processes, and sentinels for future research to unveil hidden diversity. PMID:26205828

DNA barcode goes two-dimensions: DNA QR code web server.

PubMed

Liu, Chang; Shi, Linchun; Xu, Xiaolan; Li, Huan; Xing, Hang; Liang, Dong; Jiang, Kun; Pang, Xiaohui; Song, Jingyuan; Chen, Shilin

2012-01-01

The DNA barcoding technology uses a standard region of DNA sequence for species identification and discovery. At present, "DNA barcode" actually refers to DNA sequences, which are not amenable to information storage, recognition, and retrieval. Our aim is to identify the best symbology that can represent DNA barcode sequences in practical applications. A comprehensive set of sequences for five DNA barcode markers ITS2, rbcL, matK, psbA-trnH, and CO1 was used as the test data. Fifty-three different types of one-dimensional and ten two-dimensional barcode symbologies were compared based on different criteria, such as coding capacity, compression efficiency, and error detection ability. The quick response (QR) code was found to have the largest coding capacity and relatively high compression ratio. To facilitate the further usage of QR code-based DNA barcodes, a web server was developed and is accessible at http://qrfordna.dnsalias.org. The web server allows users to retrieve the QR code for a species of interests, convert a DNA sequence to and from a QR code, and perform species identification based on local and global sequence similarities. In summary, the first comprehensive evaluation of various barcode symbologies has been carried out. The QR code has been found to be the most appropriate symbology for DNA barcode sequences. A web server has also been constructed to allow biologists to utilize QR codes in practical DNA barcoding applications.

Single molecule counting and assessment of random molecular tagging errors with transposable giga-scale error-correcting barcodes.

PubMed

Lau, Billy T; Ji, Hanlee P

2017-09-21

RNA-Seq measures gene expression by counting sequence reads belonging to unique cDNA fragments. Molecular barcodes commonly in the form of random nucleotides were recently introduced to improve gene expression measures by detecting amplification duplicates, but are susceptible to errors generated during PCR and sequencing. This results in false positive counts, leading to inaccurate transcriptome quantification especially at low input and single-cell RNA amounts where the total number of molecules present is minuscule. To address this issue, we demonstrated the systematic identification of molecular species using transposable error-correcting barcodes that are exponentially expanded to tens of billions of unique labels. We experimentally showed random-mer molecular barcodes suffer from substantial and persistent errors that are difficult to resolve. To assess our method's performance, we applied it to the analysis of known reference RNA standards. By including an inline random-mer molecular barcode, we systematically characterized the presence of sequence errors in random-mer molecular barcodes. We observed that such errors are extensive and become more dominant at low input amounts. We described the first study to use transposable molecular barcodes and its use for studying random-mer molecular barcode errors. Extensive errors found in random-mer molecular barcodes may warrant the use of error correcting barcodes for transcriptome analysis as input amounts decrease.

Digital gene expression analysis with sample multiplexing and PCR duplicate detection: A straightforward protocol.

PubMed

Rozenberg, Andrey; Leese, Florian; Weiss, Linda C; Tollrian, Ralph

2016-01-01

Tag-Seq is a high-throughput approach used for discovering SNPs and characterizing gene expression. In comparison to RNA-Seq, Tag-Seq eases data processing and allows detection of rare mRNA species using only one tag per transcript molecule. However, reduced library complexity raises the issue of PCR duplicates, which distort gene expression levels. Here we present a novel Tag-Seq protocol that uses the least biased methods for RNA library preparation combined with a novel approach for joint PCR template and sample labeling. In our protocol, input RNA is fragmented by hydrolysis, and poly(A)-bearing RNAs are selected and directly ligated to mixed DNA-RNA P5 adapters. The P5 adapters contain i5 barcodes composed of sample-specific (moderately) degenerate base regions (mDBRs), which later allow detection of PCR duplicates. The P7 adapter is attached via reverse transcription with individual i7 barcodes added during the amplification step. The resulting libraries can be sequenced on an Illumina sequencer. After sample demultiplexing and PCR duplicate removal with a free software tool we designed, the data are ready for downstream analysis. Our protocol was tested on RNA samples from predator-induced and control Daphnia microcrustaceans.

DNA Barcode Sequence Identification Incorporating Taxonomic Hierarchy and within Taxon Variability

PubMed Central

Little, Damon P.

2011-01-01

For DNA barcoding to succeed as a scientific endeavor an accurate and expeditious query sequence identification method is needed. Although a global multiple–sequence alignment can be generated for some barcoding markers (e.g. COI, rbcL), not all barcoding markers are as structurally conserved (e.g. matK). Thus, algorithms that depend on global multiple–sequence alignments are not universally applicable. Some sequence identification methods that use local pairwise alignments (e.g. BLAST) are unable to accurately differentiate between highly similar sequences and are not designed to cope with hierarchic phylogenetic relationships or within taxon variability. Here, I present a novel alignment–free sequence identification algorithm–BRONX–that accounts for observed within taxon variability and hierarchic relationships among taxa. BRONX identifies short variable segments and corresponding invariant flanking regions in reference sequences. These flanking regions are used to score variable regions in the query sequence without the production of a global multiple–sequence alignment. By incorporating observed within taxon variability into the scoring procedure, misidentifications arising from shared alleles/haplotypes are minimized. An explicit treatment of more inclusive terminals allows for separate identifications to be made for each taxonomic level and/or for user–defined terminals. BRONX performs better than all other methods when there is imperfect overlap between query and reference sequences (e.g. mini–barcode queries against a full–length barcode database). BRONX consistently produced better identifications at the genus–level for all query types. PMID:21857897

Genetic Control of Plant Root Colonization by the Biocontrol agent, Pseudomonas fluorescens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cole, Benjamin J.; Fletcher, Meghan; Waters, Jordan

Plant growth promoting rhizobacteria (PGPR) are a critical component of plant root ecosystems. PGPR promote plant growth by solubilizing inaccessible minerals, suppressing pathogenic microorganisms in the soil, and directly stimulating growth through hormone synthesis. Pseudomonas fluorescens is a well-established PGPR isolated from wheat roots that can also colonize the root system of the model plant, Arabidopsis thaliana. We have created barcoded transposon insertion mutant libraries suitable for genome-wide transposon-mediated mutagenesis followed by sequencing (TnSeq). These libraries consist of over 105 independent insertions, collectively providing loss-of-function mutants for nearly all genes in the P.fluorescens genome. Each insertion mutant can be unambiguouslymore » identified by a randomized 20 nucleotide sequence (barcode) engineered into the transposon sequence. We used these libraries in a gnotobiotic assay to examine the colonization ability of P.fluorescens on A.thaliana roots. Taking advantage of the ability to distinguish individual colonization events using barcode sequences, we assessed the timing and microbial concentration dependence of colonization of the rhizoplane niche. These data provide direct insight into the dynamics of plant root colonization in an in vivo system and define baseline parameters for the systematic identification of the bacterial genes and molecular pathways using TnSeq assays. Having determined parameters that facilitate potential colonization of roots by thousands of independent insertion mutants in a single assay, we are currently establishing a genome-wide functional map of genes required for root colonization in P.fluorescens. Importantly, the approach developed and optimized here for P.fluorescens>A.thaliana colonization will be applicable to a wide range of plant-microbe interactions, including biofuel feedstock plants and microbes known or hypothesized to impact on biofuel-relevant traits including biomass productivity and pathogen resistance.« less

DNA-encoded chemical libraries: advancing beyond conventional small-molecule libraries.

PubMed

Franzini, Raphael M; Neri, Dario; Scheuermann, Jörg

2014-04-15

DNA-encoded chemical libraries (DECLs) represent a promising tool in drug discovery. DECL technology allows the synthesis and screening of chemical libraries of unprecedented size at moderate costs. In analogy to phage-display technology, where large antibody libraries are displayed on the surface of filamentous phage and are genetically encoded in the phage genome, DECLs feature the display of individual small organic chemical moieties on DNA fragments serving as amplifiable identification barcodes. The DNA-tag facilitates the synthesis and allows the simultaneous screening of very large sets of compounds (up to billions of molecules), because the hit compounds can easily be identified and quantified by PCR-amplification of the DNA-barcode followed by high-throughput DNA sequencing. Several approaches have been used to generate DECLs, differing both in the methods used for library encoding and for the combinatorial assembly of chemical moieties. For example, DECLs can be used for fragment-based drug discovery, displaying a single molecule on DNA or two chemical moieties at the extremities of complementary DNA strands. DECLs can vary substantially in the chemical structures and the library size. While ultralarge libraries containing billions of compounds have been reported containing four or more sets of building blocks, also smaller libraries have been shown to be efficient for ligand discovery. In general, it has been found that the overall library size is a poor predictor for library performance and that the number and diversity of the building blocks are rather important indicators. Smaller libraries consisting of two to three sets of building blocks better fulfill the criteria of drug-likeness and often have higher quality. In this Account, we present advances in the DECL field from proof-of-principle studies to practical applications for drug discovery, both in industry and in academia. DECL technology can yield specific binders to a variety of target proteins and is likely to become a standard tool for pharmaceutical hit discovery, lead expansion, and Chemical Biology research. The introduction of new methodologies for library encoding and for compound synthesis in the presence of DNA is an exciting research field and will crucially contribute to the performance and the propagation of the technology.

DNA Barcoding Reveals Cryptic Diversity within Commercially Exploited Indo-Malay Carangidae (Teleosteii: Perciformes)

PubMed Central

Mat Jaafar, Tun Nurul Aimi; Taylor, Martin I.; Mohd Nor, Siti Azizah; de Bruyn, Mark; Carvalho, Gary R.

2012-01-01

Background DNA barcodes, typically focusing on the cytochrome oxidase I gene (COI) in many animals, have been used widely as a species-identification tool. The ability of DNA barcoding to distinguish species from a range of taxa and to reveal cryptic species has been well documented. Despite the wealth of DNA barcode data for fish from many temperate regions, there are relatively few available from the Southeast Asian region. Here, we target the marine fish Family Carangidae, one of the most commercially-important families from the Indo-Malay Archipelago (IMA), to produce an initial reference DNA barcode library. Methodology/Principal Findings Here, a 652 bp region of COI was sequenced for 723 individuals from 36 putative species of Family Carangidae distributed within IMA waters. Within the newly-generated dataset, three described species exhibited conspecific divergences up to ten times greater (4.32–4.82%) than mean estimates (0.24–0.39%), indicating a discrepancy with assigned morphological taxonomic identification, and the existence of cryptic species. Variability of the mitochondrial DNA COI region was compared within and among species to evaluate the COI region's suitability for species identification. The trend in range of mean K2P distances observed was generally in accordance with expectations based on taxonomic hierarchy: 0% to 4.82% between individuals within species, 0% to 16.4% between species within genera, and 8.64% to 25.39% between genera within families. The average Kimura 2-parameter (K2P) distance between individuals, between species within genera, and between genera within family were 0.37%, 10.53% and 16.56%, respectively. All described species formed monophyletic clusters in the Neighbour-joining phylogenetic tree, although three species representing complexes of six potential cryptic species were detected in Indo-Malay Carangidae; Atule mate, Selar crumenophthalmus and Seriolina nigrofasciata. Conclusion/Significance This study confirms that COI is an effective tool for species identification of Carangidae from the IMA. There were moderate levels of cryptic diversity among putative species within the central IMA. However, to explain the hypothesis of species richness in the IMA, it is necessary to sample the whole family across their broad geographic range. Such insights are helpful not only to document mechanisms driving diversification and recruitment in Carangidae, but also to provide a scientific framework for management strategies and conservation of commercially-important fisheries resources. PMID:23209586

An introduction to QR Codes: linking libraries and mobile patrons.

PubMed

Hoy, Matthew B

2011-01-01

QR codes, or "Quick Response" codes, are two-dimensional barcodes that can be scanned by mobile smartphone cameras. These codes can be used to provide fast access to URLs, telephone numbers, and short passages of text. With the rapid adoption of smartphones, librarians are able to use QR codes to promote services and help library users find materials quickly and independently. This article will explain what QR codes are, discuss how they can be used in the library, and describe issues surrounding their use. A list of resources for generating and scanning QR codes is also provided.

An Integrated Microfluidic Processor for DNA-Encoded Combinatorial Library Functional Screening

PubMed Central

2017-01-01

DNA-encoded synthesis is rekindling interest in combinatorial compound libraries for drug discovery and in technology for automated and quantitative library screening. Here, we disclose a microfluidic circuit that enables functional screens of DNA-encoded compound beads. The device carries out library bead distribution into picoliter-scale assay reagent droplets, photochemical cleavage of compound from the bead, assay incubation, laser-induced fluorescence-based assay detection, and fluorescence-activated droplet sorting to isolate hits. DNA-encoded compound beads (10-μm diameter) displaying a photocleavable positive control inhibitor pepstatin A were mixed (1920 beads, 729 encoding sequences) with negative control beads (58 000 beads, 1728 encoding sequences) and screened for cathepsin D inhibition using a biochemical enzyme activity assay. The circuit sorted 1518 hit droplets for collection following 18 min incubation over a 240 min analysis. Visual inspection of a subset of droplets (1188 droplets) yielded a 24% false discovery rate (1166 pepstatin A beads; 366 negative control beads). Using template barcoding strategies, it was possible to count hit collection beads (1863) using next-generation sequencing data. Bead-specific barcodes enabled replicate counting, and the false discovery rate was reduced to 2.6% by only considering hit-encoding sequences that were observed on >2 beads. This work represents a complete distributable small molecule discovery platform, from microfluidic miniaturized automation to ultrahigh-throughput hit deconvolution by sequencing. PMID:28199790

An Integrated Microfluidic Processor for DNA-Encoded Combinatorial Library Functional Screening.

PubMed

MacConnell, Andrew B; Price, Alexander K; Paegel, Brian M

2017-03-13

DNA-encoded synthesis is rekindling interest in combinatorial compound libraries for drug discovery and in technology for automated and quantitative library screening. Here, we disclose a microfluidic circuit that enables functional screens of DNA-encoded compound beads. The device carries out library bead distribution into picoliter-scale assay reagent droplets, photochemical cleavage of compound from the bead, assay incubation, laser-induced fluorescence-based assay detection, and fluorescence-activated droplet sorting to isolate hits. DNA-encoded compound beads (10-μm diameter) displaying a photocleavable positive control inhibitor pepstatin A were mixed (1920 beads, 729 encoding sequences) with negative control beads (58 000 beads, 1728 encoding sequences) and screened for cathepsin D inhibition using a biochemical enzyme activity assay. The circuit sorted 1518 hit droplets for collection following 18 min incubation over a 240 min analysis. Visual inspection of a subset of droplets (1188 droplets) yielded a 24% false discovery rate (1166 pepstatin A beads; 366 negative control beads). Using template barcoding strategies, it was possible to count hit collection beads (1863) using next-generation sequencing data. Bead-specific barcodes enabled replicate counting, and the false discovery rate was reduced to 2.6% by only considering hit-encoding sequences that were observed on >2 beads. This work represents a complete distributable small molecule discovery platform, from microfluidic miniaturized automation to ultrahigh-throughput hit deconvolution by sequencing.

Genome-wide mapping of autonomous promoter activity in human cells

PubMed Central

van Arensbergen, Joris; FitzPatrick, Vincent D.; de Haas, Marcel; Pagie, Ludo; Sluimer, Jasper; Bussemaker, Harmen J.; van Steensel, Bas

2017-01-01

Previous methods to systematically characterize sequence-intrinsic activity of promoters have been limited by relatively low throughput and the length of sequences that could be tested. Here we present Survey of Regulatory Elements (SuRE), a method to assay more than 108 DNA fragments, each 0.2–2kb in size, for their ability to drive transcription autonomously. In SuRE, a plasmid library is constructed of random genomic fragments upstream of a 20bp barcode and decoded by paired-end sequencing. This library is then transfected into cells and transcribed barcodes are quantified in the RNA by high throughput sequencing. When applied to the human genome, we achieved a 55-fold genome coverage, allowing us to map autonomous promoter activity genome-wide. By computational modeling we delineated subregions within promoters that are relevant for their activity. For instance, we show that antisense promoter transcription is generally dependent on the sense core promoter sequences, and that most enhancers and several families of repetitive elements act as autonomous transcription initiation sites. PMID:28024146

The Trichoptera barcode initiative: a strategy for generating a species-level Tree of Life.

PubMed

Zhou, Xin; Frandsen, Paul B; Holzenthal, Ralph W; Beet, Clare R; Bennett, Kristi R; Blahnik, Roger J; Bonada, Núria; Cartwright, David; Chuluunbat, Suvdtsetseg; Cocks, Graeme V; Collins, Gemma E; deWaard, Jeremy; Dean, John; Flint, Oliver S; Hausmann, Axel; Hendrich, Lars; Hess, Monika; Hogg, Ian D; Kondratieff, Boris C; Malicky, Hans; Milton, Megan A; Morinière, Jérôme; Morse, John C; Mwangi, François Ngera; Pauls, Steffen U; Gonzalez, María Razo; Rinne, Aki; Robinson, Jason L; Salokannel, Juha; Shackleton, Michael; Smith, Brian; Stamatakis, Alexandros; StClair, Ros; Thomas, Jessica A; Zamora-Muñoz, Carmen; Ziesmann, Tanja; Kjer, Karl M

2016-09-05

DNA barcoding was intended as a means to provide species-level identifications through associating DNA sequences from unknown specimens to those from curated reference specimens. Although barcodes were not designed for phylogenetics, they can be beneficial to the completion of the Tree of Life. The barcode database for Trichoptera is relatively comprehensive, with data from every family, approximately two-thirds of the genera, and one-third of the described species. Most Trichoptera, as with most of life's species, have never been subjected to any formal phylogenetic analysis. Here, we present a phylogeny with over 16 000 unique haplotypes as a working hypothesis that can be updated as our estimates improve. We suggest a strategy of implementing constrained tree searches, which allow larger datasets to dictate the backbone phylogeny, while the barcode data fill out the tips of the tree. We also discuss how this phylogeny could be used to focus taxonomic attention on ambiguous species boundaries and hidden biodiversity. We suggest that systematists continue to differentiate between 'Barcode Index Numbers' (BINs) and 'species' that have been formally described. Each has utility, but they are not synonyms. We highlight examples of integrative taxonomy, using both barcodes and morphology for species description.This article is part of the themed issue 'From DNA barcodes to biomes'. © 2016 The Authors.

Potential for DNA-based ID of Great Lakes fauna: Species inventories vs. barcode libraries

EPA Science Inventory

DNA-based identification of mixed-organism samples offers the potential to greatly reduce the need for resource-intensive morphological identification, which would be of value both to biotic condition assessment and non-native species early-detection monitoring. However the abil...

Using DNA barcodes to identify a bird involved in a birdstrike at a Chinese airport.

PubMed

Yang, Rong; Wu, Xiaobing; Yan, Peng; Li, Xiaoqiang

2010-10-01

One day at dusk in August, 200X, an airplane was struck by a bird at a Chinese airport (M Airport). After a careful check, some blades of the plane's engine were found to be out of shape and a few feathers and some bloodstains were found in the air intake of the engine. In order to know which species of bird was involved in the birdstrike, firstly we extracted DNA from the bloodstains; secondly, the DNA barcode (portion of COI gene) of the unknown species was amplified by PCR method; thirdly, sequence divergences (K2P differences) of the DNA barcode between the unknown species and a library of 59 common bird species distributed at the airport area were analyzed. Furthermore, a neighbor-joining (NJ) tree based on COI barcodes was created to provide graphic representation of sequence divergences among the species to confirm the identification. The result showed that red-rumped swallow (Hirundo daurica) was involved in the birdstrike incident. Some suggestions to avoid birdstrikes caused by red-rumped swallows were given to the administrative department of M Airport to ensure flying safety.

DNA barcoding implicates 23 species and four orders as potential pollinators of Chinese knotweed (Persicaria chinensis) in Peninsular Malaysia.

PubMed

Wong, M-M; Lim, C-L; Wilson, J-J

2015-08-01

Chinese knotweed (Persicaria chinensis) is of ecological and economic importance as a high-risk invasive species and a traditional medicinal herb. However, the insects associated with P. chinensis pollination have received scant attention. As a widespread invasive plant we would expect P. chinensis to be associated with a diverse group of insect pollinators, but lack of taxonomic identification capacity is an impediment to confirm this expectation. In the present study we aimed to elucidate the insect pollinators of P. chinensis in peninsular Malaysia using DNA barcoding. Forty flower visitors, representing the range of morphological diversity observed, were captured at flowers at Ulu Kali, Pahang, Malaysia. Using Automated Barcode Gap Discovery, 17 morphospecies were assigned to 23 species representing at least ten families and four orders. Using the DNA barcode library (BOLD) 30% of the species could be assigned a species name, and 70% could be assigned a genus name. The insects visiting P. chinensis were broadly similar to those previously reported as visiting Persicaria japonica, including honey bees (Apis), droneflies (Eristalis), blowflies (Lucilia) and potter wasps (Eumedes), but also included thrips and ants.

Molecular Identification of Commercialized Medicinal Plants in Southern Morocco

PubMed Central

Krüger, Åsa; Rydberg, Anders; Abbad, Abdelaziz; Björk, Lars; Martin, Gary

2012-01-01

Background Medicinal plant trade is important for local livelihoods. However, many medicinal plants are difficult to identify when they are sold as roots, powders or bark. DNA barcoding involves using a short, agreed-upon region of a genome as a unique identifier for species– ideally, as a global standard. Research Question What is the functionality, efficacy and accuracy of the use of barcoding for identifying root material, using medicinal plant roots sold by herbalists in Marrakech, Morocco, as a test dataset. Methodology In total, 111 root samples were sequenced for four proposed barcode regions rpoC1, psbA-trnH, matK and ITS. Sequences were searched against a tailored reference database of Moroccan medicinal plants and their closest relatives using BLAST and Blastclust, and through inference of RAxML phylograms of the aligned market and reference samples. Principal Findings Sequencing success was high for rpoC1, psbA-trnH, and ITS, but low for matK. Searches using rpoC1 alone resulted in a number of ambiguous identifications, indicating insufficient DNA variation for accurate species-level identification. Combining rpoC1, psbA-trnH and ITS allowed the majority of the market samples to be identified to genus level. For a minority of the market samples, the barcoding identification differed significantly from previous hypotheses based on the vernacular names. Conclusions/Significance Endemic plant species are commercialized in Marrakech. Adulteration is common and this may indicate that the products are becoming locally endangered. Nevertheless the majority of the traded roots belong to species that are common and not known to be endangered. A significant conclusion from our results is that unknown samples are more difficult to identify than earlier suggested, especially if the reference sequences were obtained from different populations. A global barcoding database should therefore contain sequences from different populations of the same species to assure the reference sequences characterize the species throughout its distributional range. PMID:22761800

Detecting very low allele fraction variants using targeted DNA sequencing and a novel molecular barcode-aware variant caller.

PubMed

Xu, Chang; Nezami Ranjbar, Mohammad R; Wu, Zhong; DiCarlo, John; Wang, Yexun

2017-01-03

Detection of DNA mutations at very low allele fractions with high accuracy will significantly improve the effectiveness of precision medicine for cancer patients. To achieve this goal through next generation sequencing, researchers need a detection method that 1) captures rare mutation-containing DNA fragments efficiently in the mix of abundant wild-type DNA; 2) sequences the DNA library extensively to deep coverage; and 3) distinguishes low level true variants from amplification and sequencing errors with high accuracy. Targeted enrichment using PCR primers provides researchers with a convenient way to achieve deep sequencing for a small, yet most relevant region using benchtop sequencers. Molecular barcoding (or indexing) provides a unique solution for reducing sequencing artifacts analytically. Although different molecular barcoding schemes have been reported in recent literature, most variant calling has been done on limited targets, using simple custom scripts. The analytical performance of barcode-aware variant calling can be significantly improved by incorporating advanced statistical models. We present here a highly efficient, simple and scalable enrichment protocol that integrates molecular barcodes in multiplex PCR amplification. In addition, we developed smCounter, an open source, generic, barcode-aware variant caller based on a Bayesian probabilistic model. smCounter was optimized and benchmarked on two independent read sets with SNVs and indels at 5 and 1% allele fractions. Variants were called with very good sensitivity and specificity within coding regions. We demonstrated that we can accurately detect somatic mutations with allele fractions as low as 1% in coding regions using our enrichment protocol and variant caller.

Diversity of Marine-Derived Fungal Cultures Exposed by DNA Barcodes: The Algorithm Matters

PubMed Central

Andreakis, Nikos; Høj, Lone; Kearns, Philip; Hall, Michael R.; Ericson, Gavin; Cobb, Rose E.; Gordon, Benjamin R.; Evans-Illidge, Elizabeth

2015-01-01

Marine fungi are an understudied group of eukaryotic microorganisms characterized by unresolved genealogies and unstable classification. Whereas DNA barcoding via the nuclear ribosomal internal transcribed spacer (ITS) provides a robust and rapid tool for fungal species delineation, accurate classification of fungi is often arduous given the large number of partial or unknown barcodes and misidentified isolates deposited in public databases. This situation is perpetuated by a paucity of cultivable fungal strains available for phylogenetic research linked to these data sets. We analyze ITS barcodes produced from a subsample (290) of 1781 cultured isolates of marine-derived fungi in the Bioresources Library located at the Australian Institute of Marine Science (AIMS). Our analysis revealed high levels of under-explored fungal diversity. The majority of isolates were ascomycetes including representatives of the subclasses Eurotiomycetidae, Hypocreomycetidae, Sordariomycetidae, Pleosporomycetidae, Dothideomycetidae, Xylariomycetidae and Saccharomycetidae. The phylum Basidiomycota was represented by isolates affiliated with the genera Tritirachium and Tilletiopsis. BLAST searches revealed 26 unknown OTUs and 50 isolates corresponding to previously uncultured, unidentified fungal clones. This study makes a significant addition to the availability of barcoded, culturable marine-derived fungi for detailed future genomic and physiological studies. We also demonstrate the influence of commonly used alignment algorithms and genetic distance measures on the accuracy and comparability of estimating Operational Taxonomic Units (OTUs) by the automatic barcode gap finder (ABGD) method. Large scale biodiversity screening programs that combine datasets using algorithmic OTU delineation pipelines need to ensure compatible algorithms have been used because the algorithm matters. PMID:26308620

VIP Barcoding: composition vector-based software for rapid species identification based on DNA barcoding.

PubMed

Fan, Long; Hui, Jerome H L; Yu, Zu Guo; Chu, Ka Hou

2014-07-01

Species identification based on short sequences of DNA markers, that is, DNA barcoding, has emerged as an integral part of modern taxonomy. However, software for the analysis of large and multilocus barcoding data sets is scarce. The Basic Local Alignment Search Tool (BLAST) is currently the fastest tool capable of handling large databases (e.g. >5000 sequences), but its accuracy is a concern and has been criticized for its local optimization. However, current more accurate software requires sequence alignment or complex calculations, which are time-consuming when dealing with large data sets during data preprocessing or during the search stage. Therefore, it is imperative to develop a practical program for both accurate and scalable species identification for DNA barcoding. In this context, we present VIP Barcoding: a user-friendly software in graphical user interface for rapid DNA barcoding. It adopts a hybrid, two-stage algorithm. First, an alignment-free composition vector (CV) method is utilized to reduce searching space by screening a reference database. The alignment-based K2P distance nearest-neighbour method is then employed to analyse the smaller data set generated in the first stage. In comparison with other software, we demonstrate that VIP Barcoding has (i) higher accuracy than Blastn and several alignment-free methods and (ii) higher scalability than alignment-based distance methods and character-based methods. These results suggest that this platform is able to deal with both large-scale and multilocus barcoding data with accuracy and can contribute to DNA barcoding for modern taxonomy. VIP Barcoding is free and available at http://msl.sls.cuhk.edu.hk/vipbarcoding/. © 2014 John Wiley & Sons Ltd.

Let's Get Physical-Or Not

ERIC Educational Resources Information Center

Kim, Ann

2005-01-01

Necessity is still the mother of invention, and physical processing is a reality for most libraries. Ergo, the inevitable development of Indigo Media and its innovative MediaPrint software, which allows distributors to overlay customized labels, logos, barcodes, and artwork onto retail labels of audiobooks, CDs, VHS tapes, DVDs, and CD-ROMs, on a…

Potential for DNA-based identification of Great Lakes fauna: Match and mismatch between taxa inventories and DNA barcode libraries

EPA Science Inventory

DNA-based identification of mixed-organism samples offers the potential to greatly reduce the need for resource-intensive morphological identification, which would be of value both to biotic condition assessment and non-native species early-detection monitoring. However, the abi...

DNA barcoding commercially important fish species of Turkey.

PubMed

Keskın, Emre; Atar, Hasan H

2013-09-01

DNA barcoding was used in the identification of 89 commercially important freshwater and marine fish species found in Turkish ichthyofauna. A total of 1765 DNA barcodes using a 654-bp-long fragment of the mitochondrial cytochrome c oxidase subunit I gene were generated for 89 commercially important freshwater and marine fish species found in Turkish ichthyofauna. These species belong to 70 genera, 40 families and 19 orders from class Actinopterygii, and all were associated with a distinct DNA barcode. Nine and 12 of the COI barcode clusters represent the first species records submitted to the BOLD and GenBank databases, respectively. All COI barcodes (except sequences of first species records) were matched with reference sequences of expected species, according to morphological identification. Average nucleotide frequencies of the data set were calculated as T = 29.7%, C = 28.2%, A = 23.6% and G = 18.6%. Average pairwise genetic distance among individuals were estimated as 0.32%, 9.62%, 17,90% and 22.40% for conspecific, congeneric, confamilial and within order, respectively. Kimura 2-parameter genetic distance values were found to increase with taxonomic level. For most of the species analysed in our data set, there is a barcoding gap, and an overlap in the barcoding gap exists for only two genera. Neighbour-joining trees were drawn based on DNA barcodes and all the specimens clustered in agreement with their taxonomic classification at species level. Results of this study supported DNA barcoding as an efficient molecular tool for a better monitoring, conservation and management of fisheries. © 2013 John Wiley & Sons Ltd.

DNA Barcode Goes Two-Dimensions: DNA QR Code Web Server

PubMed Central

Li, Huan; Xing, Hang; Liang, Dong; Jiang, Kun; Pang, Xiaohui; Song, Jingyuan; Chen, Shilin

2012-01-01

The DNA barcoding technology uses a standard region of DNA sequence for species identification and discovery. At present, “DNA barcode” actually refers to DNA sequences, which are not amenable to information storage, recognition, and retrieval. Our aim is to identify the best symbology that can represent DNA barcode sequences in practical applications. A comprehensive set of sequences for five DNA barcode markers ITS2, rbcL, matK, psbA-trnH, and CO1 was used as the test data. Fifty-three different types of one-dimensional and ten two-dimensional barcode symbologies were compared based on different criteria, such as coding capacity, compression efficiency, and error detection ability. The quick response (QR) code was found to have the largest coding capacity and relatively high compression ratio. To facilitate the further usage of QR code-based DNA barcodes, a web server was developed and is accessible at http://qrfordna.dnsalias.org. The web server allows users to retrieve the QR code for a species of interests, convert a DNA sequence to and from a QR code, and perform species identification based on local and global sequence similarities. In summary, the first comprehensive evaluation of various barcode symbologies has been carried out. The QR code has been found to be the most appropriate symbology for DNA barcode sequences. A web server has also been constructed to allow biologists to utilize QR codes in practical DNA barcoding applications. PMID:22574113

The Trichoptera barcode initiative: a strategy for generating a species-level Tree of Life

PubMed Central

Frandsen, Paul B.; Holzenthal, Ralph W.; Beet, Clare R.; Bennett, Kristi R.; Blahnik, Roger J.; Bonada, Núria; Cartwright, David; Chuluunbat, Suvdtsetseg; Cocks, Graeme V.; Collins, Gemma E.; deWaard, Jeremy; Dean, John; Flint, Oliver S.; Hausmann, Axel; Hendrich, Lars; Hess, Monika; Hogg, Ian D.; Kondratieff, Boris C.; Malicky, Hans; Milton, Megan A.; Morinière, Jérôme; Morse, John C.; Mwangi, François Ngera; Pauls, Steffen U.; Gonzalez, María Razo; Rinne, Aki; Robinson, Jason L.; Salokannel, Juha; Shackleton, Michael; Smith, Brian; Stamatakis, Alexandros; StClair, Ros; Thomas, Jessica A.; Zamora-Muñoz, Carmen; Ziesmann, Tanja

2016-01-01

DNA barcoding was intended as a means to provide species-level identifications through associating DNA sequences from unknown specimens to those from curated reference specimens. Although barcodes were not designed for phylogenetics, they can be beneficial to the completion of the Tree of Life. The barcode database for Trichoptera is relatively comprehensive, with data from every family, approximately two-thirds of the genera, and one-third of the described species. Most Trichoptera, as with most of life's species, have never been subjected to any formal phylogenetic analysis. Here, we present a phylogeny with over 16 000 unique haplotypes as a working hypothesis that can be updated as our estimates improve. We suggest a strategy of implementing constrained tree searches, which allow larger datasets to dictate the backbone phylogeny, while the barcode data fill out the tips of the tree. We also discuss how this phylogeny could be used to focus taxonomic attention on ambiguous species boundaries and hidden biodiversity. We suggest that systematists continue to differentiate between ‘Barcode Index Numbers’ (BINs) and ‘species’ that have been formally described. Each has utility, but they are not synonyms. We highlight examples of integrative taxonomy, using both barcodes and morphology for species description. This article is part of the themed issue ‘From DNA barcodes to biomes’. PMID:27481793

Bridging two scholarly islands enriches both: COI DNA barcodes for species identification versus human mitochondrial variation for the study of migrations and pathologies.

PubMed

Thaler, David S; Stoeckle, Mark Y

2016-10-01

DNA barcodes for species identification and the analysis of human mitochondrial variation have developed as independent fields even though both are based on sequences from animal mitochondria. This study finds questions within each field that can be addressed by reference to the other. DNA barcodes are based on a 648-bp segment of the mitochondrially encoded cytochrome oxidase I. From most species, this segment is the only sequence available. It is impossible to know whether it fairly represents overall mitochondrial variation. For modern humans, the entire mitochondrial genome is available from thousands of healthy individuals. SNPs in the human mitochondrial genome are evenly distributed across all protein-encoding regions arguing that COI DNA barcode is representative. Barcode variation among related species is largely based on synonymous codons. Data on human mitochondrial variation support the interpretation that most - possibly all - synonymous substitutions in mitochondria are selectively neutral. DNA barcodes confirm reports of a low variance in modern humans compared to nonhuman primates. In addition, DNA barcodes allow the comparison of modern human variance to many other extant animal species. Birds are a well-curated group in which DNA barcodes are coupled with census and geographic data. Putting modern human variation in the context of intraspecies variation among birds shows humans to be a single breeding population of average variance.

Genetic calibration of species diversity among North America's freshwater fishes.

PubMed

April, Julien; Mayden, Richard L; Hanner, Robert H; Bernatchez, Louis

2011-06-28

Freshwater ecosystems are being heavily exploited and degraded by human activities all over the world, including in North America, where fishes and fisheries are strongly affected. Despite centuries of taxonomic inquiry, problems inherent to species identification continue to hamper the conservation of North American freshwater fishes. Indeed, nearly 10% of species diversity is thought to remain undescribed. To provide an independent calibration of taxonomic uncertainty and to establish a more accessible molecular identification key for its application, we generated a standard reference library of mtDNA sequences (DNA barcodes) derived from expert-identified museum specimens for 752 North American freshwater fish species. This study demonstrates that 90% of known species can be delineated using barcodes. Moreover, it reveals numerous genetic discontinuities indicative of independently evolving lineages within described species, which points to the presence of morphologically cryptic diversity. From the 752 species analyzed, our survey flagged 138 named species that represent as many as 347 candidate species, which suggests a 28% increase in species diversity. In contrast, several species of parasitic and nonparasitic lampreys lack such discontinuity and may represent alternative life history strategies within single species. Therefore, it appears that the current North American freshwater fish taxonomy at the species level significantly conceals diversity in some groups, although artificially creating diversity in others. In addition to providing an easily accessible digital identification system, this study identifies 151 fish species for which taxonomic revision is required.

Comparative microbial diversity analyses of modern marine thrombolitic mats by barcoded pyrosequencing.

PubMed

Mobberley, Jennifer M; Ortega, Maya C; Foster, Jamie S

2012-01-01

Thrombolites are unlaminated carbonate structures that form as a result of the metabolic interactions of complex microbial mat communities. Thrombolites have a long geological history; however, little is known regarding the microbes associated with modern structures. In this study, we use a barcoded 16S rRNA gene-pyrosequencing approach coupled with morphological analysis to assess the bacterial, cyanobacterial and archaeal diversity associated with actively forming thrombolites found in Highborne Cay, Bahamas. Analyses revealed four distinct microbial mat communities referred to as black, beige, pink and button mats on the surfaces of the thrombolites. At a coarse phylogenetic resolution, the domain bacterial sequence libraries from the four mats were similar, with Proteobacteria and Cyanobacteria being the most abundant. At the finer resolution of the rRNA gene sequences, significant differences in community structure were observed, with dramatically different cyanobacterial communities. Of the four mat types, the button mats contained the highest diversity of Cyanobacteria, and were dominated by two sequence clusters with high similarity to the genus Dichothrix, an organism associated with the deposition of carbonate. Archaeal diversity was low, but varied in all mat types, and the archaeal community was predominately composed of members of the Thaumarchaeota and Euryarchaeota. The morphological and genetic data support the hypothesis that the four mat types are distinctive thrombolitic mat communities. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

How to tackle the molecular species inventory for an industrialized nation-lessons from the first phase of the German Barcode of Life initiative GBOL (2012-2015).

PubMed

Geiger, M F; Astrin, J J; Borsch, T; Burkhardt, U; Grobe, P; Hand, R; Hausmann, A; Hohberg, K; Krogmann, L; Lutz, M; Monje, C; Misof, B; Morinière, J; Müller, K; Pietsch, S; Quandt, D; Rulik, B; Scholler, M; Traunspurger, W; Haszprunar, G; Wägele, W

2016-09-01

Biodiversity loss is mainly driven by human activity. While concern grows over the fate of hot spots of biodiversity, contemporary species losses still prevail in industrialized nations. Therefore, strategies were formulated to halt or reverse the loss, driven by evidence for its value for ecosystem services. Maintenance of the latter through conservation depends on correctly identified species. To this aim, the German Federal Ministry of Education and Research is funding the GBOL project, a consortium of natural history collections, botanic gardens, and universities working on a barcode reference database for the country's fauna and flora. Several noticeable findings could be useful for future campaigns: (i) validating taxon lists to serve as a taxonomic backbone is time-consuming, but without alternative; (ii) offering financial incentives to taxonomic experts, often citizen scientists, is indispensable; (iii) completion of the libraries for widespread species enables analyses of environmental samples, but the process may not hold pace with technological advancements; (iv) discoveries of new species are among the best stories for the media; (v) a commitment to common data standards and repositories is needed, as well as transboundary cooperation between nations; (vi) after validation, all data should be published online via the BOLD to make them searchable for external users and to allow cross-checking with data from other countries.

Efficacy of the core DNA barcodes in identifying processed and poorly conserved plant materials commonly used in South African traditional medicine

PubMed Central

Mankga, Ledile T.; Yessoufou, Kowiyou; Moteetee, Annah M.; Daru, Barnabas H.; van der Bank, Michelle

2013-01-01

Abstract Medicinal plants cover a broad range of taxa, which may be phylogenetically less related but morphologically very similar. Such morphological similarity between species may lead to misidentification and inappropriate use. Also the substitution of a medicinal plant by a cheaper alternative (e.g. other non-medicinal plant species), either due to misidentification, or deliberately to cheat consumers, is an issue of growing concern. In this study, we used DNA barcoding to identify commonly used medicinal plants in South Africa. Using the core plant barcodes, matK and rbcLa, obtained from processed and poorly conserved materials sold at the muthi traditional medicine market, we tested efficacy of the barcodes in species discrimination. Based on genetic divergence, PCR amplification efficiency and BLAST algorithm, we revealed varied discriminatory potentials for the DNA barcodes. In general, the barcodes exhibited high discriminatory power, indicating their effectiveness in verifying the identity of the most common plant species traded in South African medicinal markets. BLAST algorithm successfully matched 61% of the queries against a reference database, suggesting that most of the information supplied by sellers at traditional medicinal markets in South Africa is correct. Our findings reinforce the utility of DNA barcoding technique in limiting false identification that can harm public health. PMID:24453559

Using DNA barcodes for assessing diversity in the family Hybotidae (Diptera, Empidoidea)

PubMed Central

Nagy, Zoltán T.; Sonet, Gontran; Mortelmans, Jonas; Vandewynkel, Camille; Grootaert, Patrick

2013-01-01

Abstract Empidoidea is one of the largest extant lineages of flies, but phylogenetic relationships among species of this group are poorly investigated and global diversity remains scarcely assessed. In this context, one of the most enigmatic empidoid families is Hybotidae. Within the framework of a pilot study, we barcoded 339 specimens of Old World hybotids belonging to 164 species and 22 genera (plus two Empis as outgroups) and attempted to evaluate whether patterns of intra- and interspecific divergences match the current taxonomy. We used a large sampling of diverse Hybotidae. The material came from the Palaearctic (Belgium, France, Portugal and Russian Caucasus), the Afrotropic (Democratic Republic of the Congo) and the Oriental realms (Singapore and Thailand). Thereby, we optimized lab protocols for barcoding hybotids. Although DNA barcodes generally well distinguished recognized taxa, the study also revealed a number of unexpected phenomena: e.g., undescribed taxa found within morphologically very similar or identical specimens, especially when geographic distance was large; some morphologically distinct species showed no genetic divergence; or different pattern of intraspecific divergence between populations or closely related species. Using COI sequences and simple Neighbour-Joining tree reconstructions, the monophyly of many species- and genus-level taxa was well supported, but more inclusive taxonomical levels did not receive significant bootstrap support. We conclude that in hybotids DNA barcoding might be well used to identify species, when two main constraints are considered. First, incomplete barcoding libraries hinder efficient (correct) identification. Therefore, extra efforts are needed to increase the representation of hybotids in these databases. Second, the spatial scale of sampling has to be taken into account, and especially for widespread species or species complexes with unclear taxonomy, an integrative approach has to be used to clarify species boundaries and identities. PMID:24453562

DNA barcodes from four loci provide poor resolution of taxonomic groups in the genus Crataegus.

PubMed

Zarrei, Mehdi; Talent, Nadia; Kuzmina, Maria; Lee, Jeanette; Lund, Jensen; Shipley, Paul R; Stefanović, Saša; Dickinson, Timothy A

2015-04-28

DNA barcodes can facilitate identification of organisms especially when morphological characters are limited or unobservable. To what extent this potential is realized in specific groups of plants remains to be determined. Libraries of barcode sequences from well-studied authoritatively identified plants represented by herbarium voucher specimens are needed in order for DNA barcodes to serve their intended purpose, where this is possible, and to understand the reasons behind their failure to do so, when this occurs. We evaluated four loci, widely regarded as universal DNA barcodes for plants, for their utility in hawthorn species identification. Three plastid regions, matK, rbcLa and psbA-trnH, and the internal transcribed spacer 2 (ITS2) of nuclear ribosomal DNA discriminate only some of the species of Crataegus that can be recognized on the basis of their morphology etc. This is, in part, because in Rosaceae tribe Maleae most individual plastid loci yield relatively little taxonomic resolution and, in part, because the effects of allopolyploidization have not been eliminated by concerted evolution of the ITS regions. Although individual plastid markers provided generally poor resolution of taxonomic groups in Crataegus, a few species were notable exceptions. In contrast, analyses of concatenated sequences of the 3 plastid barcode loci plus 11 additional plastid loci gave a well-resolved maternal phylogeny. In the ITS2 tree, different individuals of some species formed groups with taxonomically unrelated species. This is a sign of lineage sorting due to incomplete concerted evolution in ITS2. Incongruence between the ITS2 and plastid trees is best explained by hybridization between different lineages within the genus. In aggregate, limited between-species variation in plastid loci, hybridization and a lack of concerted evolution in ITS2 all combine to limit the utility of standard barcoding markers in Crataegus. These results have implications for authentication of hawthorn materials in natural health products. Published by Oxford University Press on behalf of the Annals of Botany Company.

DNA barcodes from four loci provide poor resolution of taxonomic groups in the genus Crataegus

PubMed Central

Zarrei, Mehdi; Talent, Nadia; Kuzmina, Maria; Lee, Jeanette; Lund, Jensen; Shipley, Paul R.; Stefanović, Saša; Dickinson, Timothy A.

2015-01-01

DNA barcodes can facilitate identification of organisms especially when morphological characters are limited or unobservable. To what extent this potential is realized in specific groups of plants remains to be determined. Libraries of barcode sequences from well-studied authoritatively identified plants represented by herbarium voucher specimens are needed in order for DNA barcodes to serve their intended purpose, where this is possible, and to understand the reasons behind their failure to do so, when this occurs. We evaluated four loci, widely regarded as universal DNA barcodes for plants, for their utility in hawthorn species identification. Three plastid regions, matK, rbcLa and psbA-trnH, and the internal transcribed spacer 2 (ITS2) of nuclear ribosomal DNA discriminate only some of the species of Crataegus that can be recognized on the basis of their morphology etc. This is, in part, because in Rosaceae tribe Maleae most individual plastid loci yield relatively little taxonomic resolution and, in part, because the effects of allopolyploidization have not been eliminated by concerted evolution of the ITS regions. Although individual plastid markers provided generally poor resolution of taxonomic groups in Crataegus, a few species were notable exceptions. In contrast, analyses of concatenated sequences of the 3 plastid barcode loci plus 11 additional plastid loci gave a well-resolved maternal phylogeny. In the ITS2 tree, different individuals of some species formed groups with taxonomically unrelated species. This is a sign of lineage sorting due to incomplete concerted evolution in ITS2. Incongruence between the ITS2 and plastid trees is best explained by hybridization between different lineages within the genus. In aggregate, limited between-species variation in plastid loci, hybridization and a lack of concerted evolution in ITS2 all combine to limit the utility of standard barcoding markers in Crataegus. These results have implications for authentication of hawthorn materials in natural health products. PMID:25926325

Joining Inventory by Parataxonomists with DNA Barcoding of a Large Complex Tropical Conserved Wildland in Northwestern Costa Rica

PubMed Central

Janzen, Daniel H.; Hallwachs, Winnie

2011-01-01

Background The many components of conservation through biodiversity development of a large complex tropical wildland, Area de Conservacion Guanacaste (ACG), thrive on knowing what is its biodiversity and natural history. For 32 years a growing team of Costa Rican parataxonomists has conducted biodiversity inventory of ACG caterpillars, their food plants, and their parasitoids. In 2003, DNA barcoding was added to the inventory process. Methodology/Principal Findings We describe some of the salient consequences for the parataxonomists of barcoding becoming part of a field biodiversity inventory process that has centuries of tradition. From the barcoding results, the parataxonomists, as well as other downstream users, gain a more fine-scale and greater understanding of the specimens they find, rear, photograph, database and deliver. The parataxonomists also need to adjust to collecting more specimens of what appear to be the “same species” – cryptic species that cannot be distinguished by eye or even food plant alone – while having to work with the name changes and taxonomic uncertainty that comes with discovering that what looked like one species may be many. Conclusions/Significance These career parataxonomists, despite their lack of formal higher education, have proven very capable of absorbing and working around the additional complexity and requirements for accuracy and detail that are generated by adding barcoding to the field base of the ACG inventory. In the process, they have also gained a greater understanding of the fine details of phylogeny, relatedness, evolution, and species-packing in their own tropical complex ecosytems. There is no reason to view DNA barcoding as incompatible in any way with tropical biodiversity inventory as conducted by parataxonomists. Their year-round on-site inventory effort lends itself well to the sampling patterns and sample sizes needed to build a thorough barcode library. Furthermore, the biological understanding that comes with barcoding increases the scientific penetrance of biodiversity information, DNA understanding, evolution, and ecology into the communities in which the parataxonomists and their families are resident. PMID:21857894

The HTS barcode checker pipeline, a tool for automated detection of illegally traded species from high-throughput sequencing data.

PubMed

Lammers, Youri; Peelen, Tamara; Vos, Rutger A; Gravendeel, Barbara

2014-02-06

Mixtures of internationally traded organic substances can contain parts of species protected by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). These mixtures often raise the suspicion of border control and customs offices, which can lead to confiscation, for example in the case of Traditional Chinese medicines (TCMs). High-throughput sequencing of DNA barcoding markers obtained from such samples provides insight into species constituents of mixtures, but manual cross-referencing of results against the CITES appendices is labor intensive. Matching DNA barcodes against NCBI GenBank using BLAST may yield misleading results both as false positives, due to incorrectly annotated sequences, and false negatives, due to spurious taxonomic re-assignment. Incongruence between the taxonomies of CITES and NCBI GenBank can result in erroneous estimates of illegal trade. The HTS barcode checker pipeline is an application for automated processing of sets of 'next generation' barcode sequences to determine whether these contain DNA barcodes obtained from species listed on the CITES appendices. This analytical pipeline builds upon and extends existing open-source applications for BLAST matching against the NCBI GenBank reference database and for taxonomic name reconciliation. In a single operation, reads are converted into taxonomic identifications matched with names on the CITES appendices. By inclusion of a blacklist and additional names databases, the HTS barcode checker pipeline prevents false positives and resolves taxonomic heterogeneity. The HTS barcode checker pipeline can detect and correctly identify DNA barcodes of CITES-protected species from reads obtained from TCM samples in just a few minutes. The pipeline facilitates and improves molecular monitoring of trade in endangered species, and can aid in safeguarding these species from extinction in the wild. The HTS barcode checker pipeline is available at https://github.com/naturalis/HTS-barcode-checker.

The HTS barcode checker pipeline, a tool for automated detection of illegally traded species from high-throughput sequencing data

PubMed Central

2014-01-01

Background Mixtures of internationally traded organic substances can contain parts of species protected by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). These mixtures often raise the suspicion of border control and customs offices, which can lead to confiscation, for example in the case of Traditional Chinese medicines (TCMs). High-throughput sequencing of DNA barcoding markers obtained from such samples provides insight into species constituents of mixtures, but manual cross-referencing of results against the CITES appendices is labor intensive. Matching DNA barcodes against NCBI GenBank using BLAST may yield misleading results both as false positives, due to incorrectly annotated sequences, and false negatives, due to spurious taxonomic re-assignment. Incongruence between the taxonomies of CITES and NCBI GenBank can result in erroneous estimates of illegal trade. Results The HTS barcode checker pipeline is an application for automated processing of sets of 'next generation’ barcode sequences to determine whether these contain DNA barcodes obtained from species listed on the CITES appendices. This analytical pipeline builds upon and extends existing open-source applications for BLAST matching against the NCBI GenBank reference database and for taxonomic name reconciliation. In a single operation, reads are converted into taxonomic identifications matched with names on the CITES appendices. By inclusion of a blacklist and additional names databases, the HTS barcode checker pipeline prevents false positives and resolves taxonomic heterogeneity. Conclusions The HTS barcode checker pipeline can detect and correctly identify DNA barcodes of CITES-protected species from reads obtained from TCM samples in just a few minutes. The pipeline facilitates and improves molecular monitoring of trade in endangered species, and can aid in safeguarding these species from extinction in the wild. The HTS barcode checker pipeline is available at https://github.com/naturalis/HTS-barcode-checker. PMID:24502833

A universal next generation sequencing protocol to generate non-infectious barcoded cDNA libraries from high containment RNA viruses

USDA-ARS?s Scientific Manuscript database

Several biosafety level (BSL)-3/4 pathogens are high consequence, single-stranded RNA viruses and their genomes, when introduced into permissive cells, are infectious. Moreover many of these viruses are Select Agents (SAs), and their genomes are also considered SAs. For this reason cDNAs and/or th...

Molluscs for Sale: Assessment of Freshwater Gastropods and Bivalves in the Ornamental Pet Trade.

PubMed

Ng, Ting Hui; Tan, Siong Kiat; Wong, Wing Hing; Meier, Rudolf; Chan, Sow-Yan; Tan, Heok Hui; Yeo, Darren C J

2016-01-01

The ornamental pet trade is often considered a key culprit for conservation problems such as the introduction of invasive species (including infectious diseases) and overharvesting of rare species. Here, we present the first assessment of the biodiversity of freshwater molluscs in the ornamental pet trade in Singapore, one of the most important global hubs of the ornamental aquarium trade, and discuss associated conservation concerns. We recorded freshwater molluscs from ornamental pet shops and major exporters including non-ornamental species (e.g., hitchhikers, molluscs sold as fish feed). We recorded an unexpectedly high diversity-59 species-of freshwater bivalves and gastropods, with the majority (38 species or 64%) being from the Oriental region. In addition to morphological examination, we sequenced the DNA barcode region of mitochondrial CO1 and 16S genes to provide molecular data for the confirmation of the identification and for future re-identification. DNA barcodes were obtained for 50 species, and all but four were separated by > 3% uncorrected pairwise distances. The trade has been considered a main introduction pathway for non-native species to Singapore, and we found that out of 15 species in the trade as well as in the wild in Singapore, 12 are either introduced or of unknown origin, representing almost half of the known non-native freshwater molluscs in Singapore. Particularly prevalent are non-ornamental species: six hitchhikers on aquarium plants and six species sold as fish feed. We found that a quarter of the trade species have a history of introduction, which includes 11 known or potentially invasive species. We conclude that potential overharvesting is difficult to assess because only half of the trade species have been treated by IUCN. Of these, 21 species are of Least Concern and three are Data Deficient. Our checklist, with accompanying DNA barcodes, images, and museum vouchers, provides an important reference library for future monitoring, and constitutes a step toward creating a more sustainable ornamental pet trade.

Molluscs for Sale: Assessment of Freshwater Gastropods and Bivalves in the Ornamental Pet Trade

PubMed Central

Tan, Siong Kiat; Wong, Wing Hing; Meier, Rudolf; Chan, Sow-Yan; Tan, Heok Hui; Yeo, Darren C. J.

2016-01-01

The ornamental pet trade is often considered a key culprit for conservation problems such as the introduction of invasive species (including infectious diseases) and overharvesting of rare species. Here, we present the first assessment of the biodiversity of freshwater molluscs in the ornamental pet trade in Singapore, one of the most important global hubs of the ornamental aquarium trade, and discuss associated conservation concerns. We recorded freshwater molluscs from ornamental pet shops and major exporters including non-ornamental species (e.g., hitchhikers, molluscs sold as fish feed). We recorded an unexpectedly high diversity—59 species—of freshwater bivalves and gastropods, with the majority (38 species or 64%) being from the Oriental region. In addition to morphological examination, we sequenced the DNA barcode region of mitochondrial CO1 and 16S genes to provide molecular data for the confirmation of the identification and for future re-identification. DNA barcodes were obtained for 50 species, and all but four were separated by > 3% uncorrected pairwise distances. The trade has been considered a main introduction pathway for non-native species to Singapore, and we found that out of 15 species in the trade as well as in the wild in Singapore, 12 are either introduced or of unknown origin, representing almost half of the known non-native freshwater molluscs in Singapore. Particularly prevalent are non-ornamental species: six hitchhikers on aquarium plants and six species sold as fish feed. We found that a quarter of the trade species have a history of introduction, which includes 11 known or potentially invasive species. We conclude that potential overharvesting is difficult to assess because only half of the trade species have been treated by IUCN. Of these, 21 species are of Least Concern and three are Data Deficient. Our checklist, with accompanying DNA barcodes, images, and museum vouchers, provides an important reference library for future monitoring, and constitutes a step toward creating a more sustainable ornamental pet trade. PMID:27525660

Detection of plant-based adulterants in turmeric powder using DNA barcoding.

PubMed

Parvathy, V A; Swetha, V P; Sheeja, T E; Sasikumar, B

2015-01-01

In its powdered form, turmeric [Curcuma longa L. (Zingiberaceae)], a spice of medical importance, is often adulterated lowering its quality. The study sought to detect plant-based adulterants in traded turmeric powder using DNA barcoding. Accessions of Curcuma longa L., Curcuma zedoaria Rosc. (Zingiberaceae), and cassava starch served as reference samples. Three barcoding loci, namely ITS, rbcL, and matK, were used for PCR amplification of the reference samples and commercial samples representing 10 different companies. PCR success rate, sequencing efficiency, occurrence of SNPs, and BLAST analysis were used to assess the potential of the barcoding loci in authenticating the traded samples of turmeric. The PCR and sequencing success of the loci rbcL and ITS were found to be 100%, whereas matK showed no amplification. ITS proved to be the ideal locus because it showed greater variability than rbcL in discriminating the Curcuma species. The presence of C. zedoaria could be detected in one of the samples whereas cassava starch, wheat, barley, and rye in other two samples although the label claimed nothing other than turmeric powder in the samples. Unlabeled materials in turmeric powder are considered as adulterants or fillers, added to increase the bulk weight and starch content of the commodity for economic gains. These adulterants pose potential health hazards to consumers who are allergic to these plants, lowering the product's medicinal value and belying the claim that the product is gluten free. The study proved DNA barcoding as an efficient tool for testing the integrity and the authenticity of commercial products of turmeric.

Molecular-based rapid inventories of sympatric diversity: a comparison of DNA barcode clustering methods applied to geography-based vs clade-based sampling of amphibians.

PubMed

Paz, Andrea; Crawford, Andrew J

2012-11-01

Molecular markers offer a universal source of data for quantifying biodiversity. DNA barcoding uses a standardized genetic marker and a curated reference database to identify known species and to reveal cryptic diversity within wellsampled clades. Rapid biological inventories, e.g. rapid assessment programs (RAPs), unlike most barcoding campaigns, are focused on particular geographic localities rather than on clades. Because of the potentially sparse phylogenetic sampling, the addition of DNA barcoding to RAPs may present a greater challenge for the identification of named species or for revealing cryptic diversity. In this article we evaluate the use of DNA barcoding for quantifying lineage diversity within a single sampling site as compared to clade-based sampling, and present examples from amphibians. We compared algorithms for identifying DNA barcode clusters (e.g. species, cryptic species or Evolutionary Significant Units) using previously published DNA barcode data obtained from geography-based sampling at a site in Central Panama, and from clade-based sampling in Madagascar. We found that clustering algorithms based on genetic distance performed similarly on sympatric as well as clade-based barcode data, while a promising coalescent-based method performed poorly on sympatric data. The various clustering algorithms were also compared in terms of speed and software implementation. Although each method has its shortcomings in certain contexts, we recommend the use of the ABGD method, which not only performs fairly well under either sampling method, but does so in a few seconds and with a user-friendly Web interface.

Machine Learned Replacement of N-Labels for Basecalled Sequences in DNA Barcoding.

PubMed

Ma, Eddie Y T; Ratnasingham, Sujeevan; Kremer, Stefan C

2018-01-01

This study presents a machine learning method that increases the number of identified bases in Sanger Sequencing. The system post-processes a KB basecalled chromatogram. It selects a recoverable subset of N-labels in the KB-called chromatogram to replace with basecalls (A,C,G,T). An N-label correction is defined given an additional read of the same sequence, and a human finished sequence. Corrections are added to the dataset when an alignment determines the additional read and human agree on the identity of the N-label. KB must also rate the replacement with quality value of in the additional read. Corrections are only available during system training. Developing the system, nearly 850,000 N-labels are obtained from Barcode of Life Datasystems, the premier database of genetic markers called DNA Barcodes. Increasing the number of correct bases improves reference sequence reliability, increases sequence identification accuracy, and assures analysis correctness. Keeping with barcoding standards, our system maintains an error rate of percent. Our system only applies corrections when it estimates low rate of error. Tested on this data, our automation selects and recovers: 79 percent of N-labels from COI (animal barcode); 80 percent from matK and rbcL (plant barcodes); and 58 percent from non-protein-coding sequences (across eukaryotes).

DNA Barcoding for Identification of ‘Candidatus Phytoplasmas’ Using a Fragment of the Elongation Factor Tu Gene

PubMed Central

Makarova, Olga; Contaldo, Nicoletta; Paltrinieri, Samanta; Kawube, Geofrey; Bertaccini, Assunta; Nicolaisen, Mogens

2012-01-01

Background Phytoplasmas are bacterial phytopathogens responsible for significant losses in agricultural production worldwide. Several molecular markers are available for identification of groups or strains of phytoplasmas. However, they often cannot be used for identification of phytoplasmas from different groups simultaneously or are too long for routine diagnostics. DNA barcoding recently emerged as a convenient tool for species identification. Here, the development of a universal DNA barcode based on the elongation factor Tu (tuf) gene for phytoplasma identification is reported. Methodology/Principal Findings We designed a new set of primers and amplified a 420–444 bp fragment of tuf from all 91 phytoplasmas strains tested (16S rRNA groups -I through -VII, -IX through -XII, -XV, and -XX). Comparison of NJ trees constructed from the tuf barcode and a 1.2 kbp fragment of the 16S ribosomal gene revealed that the tuf tree is highly congruent with the 16S rRNA tree and had higher inter- and intra- group sequence divergence. Mean K2P inter−/intra- group divergences of the tuf barcode did not overlap and had approximately one order of magnitude difference for most groups, suggesting the presence of a DNA barcoding gap. The use of the tuf barcode allowed separation of main ribosomal groups and most of their subgroups. Phytoplasma tuf barcodes were deposited in the NCBI GenBank and Q-bank databases. Conclusions/Significance This study demonstrates that DNA barcoding principles can be applied for identification of phytoplasmas. Our findings suggest that the tuf barcode performs as well or better than a 1.2 kbp fragment of the 16S rRNA gene and thus provides an easy procedure for phytoplasma identification. The obtained sequences were used to create a publicly available reference database that can be used by plant health services and researchers for online phytoplasma identification. PMID:23272216

A new method for species identification via protein-coding and non-coding DNA barcodes by combining machine learning with bioinformatic methods.

PubMed

Zhang, Ai-bing; Feng, Jie; Ward, Robert D; Wan, Ping; Gao, Qiang; Wu, Jun; Zhao, Wei-zhong

2012-01-01

Species identification via DNA barcodes is contributing greatly to current bioinventory efforts. The initial, and widely accepted, proposal was to use the protein-coding cytochrome c oxidase subunit I (COI) region as the standard barcode for animals, but recently non-coding internal transcribed spacer (ITS) genes have been proposed as candidate barcodes for both animals and plants. However, achieving a robust alignment for non-coding regions can be problematic. Here we propose two new methods (DV-RBF and FJ-RBF) to address this issue for species assignment by both coding and non-coding sequences that take advantage of the power of machine learning and bioinformatics. We demonstrate the value of the new methods with four empirical datasets, two representing typical protein-coding COI barcode datasets (neotropical bats and marine fish) and two representing non-coding ITS barcodes (rust fungi and brown algae). Using two random sub-sampling approaches, we demonstrate that the new methods significantly outperformed existing Neighbor-joining (NJ) and Maximum likelihood (ML) methods for both coding and non-coding barcodes when there was complete species coverage in the reference dataset. The new methods also out-performed NJ and ML methods for non-coding sequences in circumstances of potentially incomplete species coverage, although then the NJ and ML methods performed slightly better than the new methods for protein-coding barcodes. A 100% success rate of species identification was achieved with the two new methods for 4,122 bat queries and 5,134 fish queries using COI barcodes, with 95% confidence intervals (CI) of 99.75-100%. The new methods also obtained a 96.29% success rate (95%CI: 91.62-98.40%) for 484 rust fungi queries and a 98.50% success rate (95%CI: 96.60-99.37%) for 1094 brown algae queries, both using ITS barcodes.

Identification of cephalopod species from the North and Baltic Seas using morphology, COI and 18S rDNA sequences

NASA Astrophysics Data System (ADS)

Gebhardt, Katharina; Knebelsberger, Thomas

2015-09-01

We morphologically analyzed 79 cephalopod specimens from the North and Baltic Seas belonging to 13 separate species. Another 29 specimens showed morphological features of either Alloteuthis mediaor Alloteuthis subulata or were found to be in between. Reliable identification features to distinguish between A. media and A. subulata are currently not available. The analysis of the DNA barcoding region of the COI gene revealed intraspecific distances (uncorrected p) ranging from 0 to 2.13 % (average 0.1 %) and interspecific distances between 3.31 and 22 % (average 15.52 %). All species formed monophyletic clusters in a neighbor-joining analysis and were supported by bootstrap values of ≥99 %. All COI haplotypes belonging to the 29 Alloteuthis specimens were grouped in one cluster. Neither COI nor 18S rDNA sequences helped to distinguish between the different Alloteuthis morphotypes. For species identification purposes, we recommend the use of COI, as it showed higher bootstrap support of species clusters and less amplification and sequencing failure compared to 18S. Our data strongly support the assumption that the genus Alloteuthis is only represented by a single species, at least in the North Sea. It remained unclear whether this species is A. subulata or A. media. All COI sequences including important metadata were uploaded to the Barcode of Life Data Systems and can be used as reference library for the molecular identification of more than 50 % of the cephalopod fauna known from the North and Baltic Seas.

Genetic calibration of species diversity among North America's freshwater fishes

PubMed Central

April, Julien; Mayden, Richard L.; Hanner, Robert H.; Bernatchez, Louis

2011-01-01

Freshwater ecosystems are being heavily exploited and degraded by human activities all over the world, including in North America, where fishes and fisheries are strongly affected. Despite centuries of taxonomic inquiry, problems inherent to species identification continue to hamper the conservation of North American freshwater fishes. Indeed, nearly 10% of species diversity is thought to remain undescribed. To provide an independent calibration of taxonomic uncertainty and to establish a more accessible molecular identification key for its application, we generated a standard reference library of mtDNA sequences (DNA barcodes) derived from expert-identified museum specimens for 752 North American freshwater fish species. This study demonstrates that 90% of known species can be delineated using barcodes. Moreover, it reveals numerous genetic discontinuities indicative of independently evolving lineages within described species, which points to the presence of morphologically cryptic diversity. From the 752 species analyzed, our survey flagged 138 named species that represent as many as 347 candidate species, which suggests a 28% increase in species diversity. In contrast, several species of parasitic and nonparasitic lampreys lack such discontinuity and may represent alternative life history strategies within single species. Therefore, it appears that the current North American freshwater fish taxonomy at the species level significantly conceals diversity in some groups, although artificially creating diversity in others. In addition to providing an easily accessible digital identification system, this study identifies 151 fish species for which taxonomic revision is required. PMID:21670289

Barcoded NS31/AML2 primers for sequencing of arbuscular mycorrhizal communities in environmental samples1

PubMed Central

Morgan, Benjamin S. T.; Egerton-Warburton, Louise M.

2017-01-01

Premise of the study: Arbuscular mycorrhizal fungi (AMF) are globally important root symbioses that enhance plant growth and nutrition and influence ecosystem structure and function. To better characterize levels of AMF diversity relevant to ecosystem function, deeper sequencing depth in environmental samples is needed. In this study, Illumina barcoded primers and a bioinformatics pipeline were developed and applied to study AMF diversity and community structure in environmental samples. Methods: Libraries of small subunit ribosomal RNA fragment amplicons were amplified from environmental DNA using a single-step PCR reaction with barcoded NS31/AML2 primers. Amplicons were sequenced on an Illumina MiSeq sequencer using version 2, 2 × 250-bp paired-end chemistry, and analyzed using QIIME and RDP Classifier. Results: Sequencing captured 196 to 6416 operational taxonomic units (OTUs; depending on clustering parameters) representing nine AMF genera. Regardless of clustering parameters, ∼20 OTUs dominated AMF communities (78–87% reads) with the remaining reads distributed among other OTUs. Analyses also showed significant biogeographic differences in AMF communities and that community composition could be linked to specific edaphic factors. Discussion: Barcoded NS31/AML2 primers and Illumina MiSeq sequencing provide a powerful approach to address AMF diversity and variations in fungal assemblages across host plants, ecosystems, and responses to environmental drivers including global change. PMID:28924511

Identification of Belgian mosquito species (Diptera: Culicidae) by DNA barcoding.

PubMed

Versteirt, V; Nagy, Z T; Roelants, P; Denis, L; Breman, F C; Damiens, D; Dekoninck, W; Backeljau, T; Coosemans, M; Van Bortel, W

2015-03-01

Since its introduction in 2003, DNA barcoding has proven to be a promising method for the identification of many taxa, including mosquitoes (Diptera: Culicidae). Many mosquito species are potential vectors of pathogens, and correct identification in all life stages is essential for effective mosquito monitoring and control. To use DNA barcoding for species identification, a reliable and comprehensive reference database of verified DNA sequences is required. Hence, DNA sequence diversity of mosquitoes in Belgium was assessed using a 658 bp fragment of the mitochondrial cytochrome oxidase I (COI) gene, and a reference data set was established. Most species appeared as well-supported clusters. Intraspecific Kimura 2-parameter (K2P) distances averaged 0.7%, and the maximum observed K2P distance was 6.2% for Aedes koreicus. A small overlap between intra- and interspecific K2P distances for congeneric sequences was observed. Overall, the identification success using best match and the best close match criteria were high, that is above 98%. No clear genetic division was found between the closely related species Aedes annulipes and Aedes cantans, which can be confused using morphological identification only. The members of the Anopheles maculipennis complex, that is Anopheles maculipennis s.s. and An. messeae, were weakly supported as monophyletic taxa. This study showed that DNA barcoding offers a reliable framework for mosquito species identification in Belgium except for some closely related species. © 2014 John Wiley & Sons Ltd.

Beyond the Colours: Discovering Hidden Diversity in the Nymphalidae of the Yucatan Peninsula in Mexico through DNA Barcoding

PubMed Central

Prado, Blanca R.; Pozo, Carmen; Valdez-Moreno, Martha; Hebert, Paul D. N.

2011-01-01

Background Recent studies have demonstrated the utility of DNA barcoding in the discovery of overlooked species and in the connection of immature and adult stages. In this study, we use DNA barcoding to examine diversity patterns in 121 species of Nymphalidae from the Yucatan Peninsula in Mexico. Our results suggest the presence of cryptic species in 8 of these 121 taxa. As well, the reference database derived from the analysis of adult specimens allowed the identification of nymphalid caterpillars providing new details on host plant use. Methodology/Principal Findings We gathered DNA barcode sequences from 857 adult Nymphalidae representing 121 different species. This total includes four species (Adelpha iphiclus, Adelpha malea, Hamadryas iphtime and Taygetis laches) that were initially overlooked because of their close morphological similarity to other species. The barcode results showed that each of the 121 species possessed a diagnostic array of barcode sequences. In addition, there was evidence of cryptic taxa; seven species included two barcode clusters showing more than 2% sequence divergence while one species included three clusters. All 71 nymphalid caterpillars were identified to a species level by their sequence congruence to adult sequences. These caterpillars represented 16 species, and included Hamadryas julitta, an endemic species from the Yucatan Peninsula whose larval stages and host plant (Dalechampia schottii, also endemic to the Yucatan Peninsula) were previously unknown. Conclusions/Significance This investigation has revealed overlooked species in a well-studied museum collection of nymphalid butterflies and suggests that there is a substantial incidence of cryptic species that await full characterization. The utility of barcoding in the rapid identification of caterpillars also promises to accelerate the assembly of information on life histories, a particularly important advance for hyperdiverse tropical insect assemblages. PMID:22132140

Mini-DNA barcode in identification of the ornamental fish: A case study from Northeast India.

PubMed

Dhar, Bishal; Ghosh, Sankar Kumar

2017-09-05

The ornamental fishes were exported under the trade names or generic names, thus creating problems in species identification. In this regard, DNA barcoding could effectively elucidate the actual species status. However, the problem arises if the specimen is having taxonomic disputes, falsified by trade/generic names, etc., On the other hand, barcoding the archival museum specimens would be of greater benefit to address such issues as it would create firm, error-free reference database for rapid identification of any species. This can be achieved only by generating short sequences as DNA from chemically preserved are mostly degraded. Here we aimed to identify a short stretch of informative sites within the full-length barcode segment, capable of delineating diverse group of ornamental fish species, commonly traded from NE India. We analyzed 287 full-length barcode sequences from the major fish orders and compared the interspecific K2P distance with nucleotide substitutions patterns and found a strong correlation of interspecies distance with transversions (0.95, p<0.001). We, therefore, proposed a short stretch of 171bp (transversion rich) segment as mini-barcode. The proposed segment was compared with the full-length barcodes and found to delineate the species effectively. Successful PCR amplification and sequencing of the 171bp segment using designed primers for different orders validated it as mini-barcodes for ornamental fishes. Thus, our findings would be helpful in strengthening the global database with the sequence of archived fish species as well as an effective identification tool of the traded ornamental fish species, as a less time consuming, cost effective field-based application. Copyright © 2017 Elsevier B.V. All rights reserved.

Identifying Canadian Freshwater Fishes through DNA Barcodes

PubMed Central

Hubert, Nicolas; Hanner, Robert; Holm, Erling; Mandrak, Nicholas E.; Taylor, Eric; Burridge, Mary; Watkinson, Douglas; Dumont, Pierre; Curry, Allen; Bentzen, Paul; Zhang, Junbin; April, Julien; Bernatchez, Louis

2008-01-01

Background DNA barcoding aims to provide an efficient method for species-level identifications using an array of species specific molecular tags derived from the 5′ region of the mitochondrial cytochrome c oxidase I (COI) gene. The efficiency of the method hinges on the degree of sequence divergence among species and species-level identifications are relatively straightforward when the average genetic distance among individuals within a species does not exceed the average genetic distance between sister species. Fishes constitute a highly diverse group of vertebrates that exhibit deep phenotypic changes during development. In this context, the identification of fish species is challenging and DNA barcoding provide new perspectives in ecology and systematics of fishes. Here we examined the degree to which DNA barcoding discriminate freshwater fish species from the well-known Canadian fauna, which currently encompasses nearly 200 species, some which are of high economic value like salmons and sturgeons. Methodology/Principal Findings We bi-directionally sequenced the standard 652 bp “barcode” region of COI for 1360 individuals belonging to 190 of the 203 Canadian freshwater fish species (95%). Most species were represented by multiple individuals (7.6 on average), the majority of which were retained as voucher specimens. The average genetic distance was 27 fold higher between species than within species, as K2P distance estimates averaged 8.3% among congeners and only 0.3% among concpecifics. However, shared polymorphism between sister-species was detected in 15 species (8% of the cases). The distribution of K2P distance between individuals and species overlapped and identifications were only possible to species group using DNA barcodes in these cases. Conversely, deep hidden genetic divergence was revealed within two species, suggesting the presence of cryptic species. Conclusions/Significance The present study evidenced that freshwater fish species can be efficiently identified through the use of DNA barcoding, especially the species complex of small-sized species, and that the present COI library can be used for subsequent applications in ecology and systematics. PMID:22423312

DNA-barcoding of forensically important blow flies (Diptera: Calliphoridae) in the Caribbean Region

PubMed Central

Agnarsson, Ingi

2017-01-01

Correct identification of forensically important insects, such as flies in the family Calliphoridae, is a crucial step for them to be used as evidence in legal investigations. Traditional identification based on morphology has been effective, but has some limitations when it comes to identifying immature stages of certain species. DNA-barcoding, using COI, has demonstrated potential for rapid and accurate identification of Calliphoridae, however, this gene does not reliably distinguish among some recently diverged species, raising questions about its use for delimitation of species of forensic importance. To facilitate DNA based identification of Calliphoridae in the Caribbean we developed a vouchered reference collection from across the region, and a DNA sequence database, and further added the nuclear ITS2 as a second marker to increase accuracy of identification through barcoding. We morphologically identified freshly collected specimens, did phylogenetic analyses and employed several species delimitation methods for a total of 468 individuals representing 19 described species. Our results show that combination of COI + ITS2 genes yields more accurate identification and diagnoses, and better agreement with morphological data, than the mitochondrial barcodes alone. All of our results from independent and concatenated trees and most of the species delimitation methods yield considerably higher diversity estimates than the distance based approach and morphology. Molecular data support at least 24 distinct clades within Calliphoridae in this study, recovering substantial geographic variation for Lucilia eximia, Lucilia retroversa, Lucilia rica and Chloroprocta idioidea, probably indicating several cryptic species. In sum, our study demonstrates the importance of employing a second nuclear marker for barcoding analyses and species delimitation of calliphorids, and the power of molecular data in combination with a complete reference database to enable identification of taxonomically and geographically diverse insects of forensic importance. PMID:28761780

DNA barcoding for biosecurity: case studies from the UK plant protection program.

PubMed

Hodgetts, Jennifer; Ostojá-Starzewski, Jozef C; Prior, Thomas; Lawson, Rebecca; Hall, Jayne; Boonham, Neil

2016-11-01

Since its conception, DNA barcoding has seen a rapid uptake within the research community. Nevertheless, as with many new scientific tools, progression towards the point of routine deployment within diagnostic laboratories has been slow. In this paper, we discuss the application of DNA barcoding in the Defra plant health diagnostic laboratories, where DNA barcoding is used primarily for the identification of invertebrate pests. We present a series of case studies that demonstrate the successful application of DNA barcoding but also reveal some potential limitations to expanded use. The regulated plant pest, Bursephalenchus xylophilus, and one of its vectors, Monochamus alternatus, were found in dining chairs. Some traded wood products are potentially high risk, allowing the movement of longhorn beetles; Trichoferus campestris, Leptura quadrifasciata, and Trichoferus holosericeus were found in a wooden cutlery tray, a railway sleeper, and a dining chair, respectively. An outbreak of Meloidogyne fallax was identified in Allium ampeloprasum and in three weed species. Reference sequences for UK native psyllids were generated to enable the development of rapid diagnostics to be used for monitoring following the release of Aphalara itadori as a biological control agent for Fallopia japonica.

An aptamer-based bio-barcode assay with isothermal recombinase polymerase amplification for cytochrome-c detection and anti-cancer drug screening.

PubMed

Loo, Jacky F C; Lau, P M; Ho, H P; Kong, S K

2013-10-15

Based on a recently reported ultra-sensitive bio-barcode (BBC) assay, we have developed an aptamer-based bio-barcode (ABC) alternative to detect a cell death marker cytochrome-c (Cyto-c) and its subsequent application to screen anti-cancer drugs. Aptamer is a short single-stranded DNA selected from a synthetic DNA library by virtue of its high binding affinity and specificity to its target based on its unique 3D structure from the nucleotide sequence after folding. In the BBC assay, an antigen (Ag) in analytes is captured by a micro-magnetic particle (MMP) coated with capturing antibodies (Abs). Gold nanoparticles (NPs) with another recognition Ab against the same target and hundreds of identical DNA molecules of known sequence are subsequently added to allow the formation of sandwich structures ([MMP-Ab1]-Ag-[Ab2-NP-DNA]). After isolating the sandwiches by a magnetic field, the DNAs hybridized to their complementary DNAs covalently bound on the NPs are released from the sandwiches after heating. Acting as an Ag identification tag, these bio-barcode DNAs with known DNA sequence are then amplified by polymerase chain reaction (PCR) and detected by fluorescence. In our ABC assay, we employed a Cyto-c-specific aptamer to substitute both the recognition Ab and barcode DNAs on the NPs in the BBC assay; and a novel isothermal recombinase polymerase amplification for the time-consuming PCR. The detection limit of our ABC assay for the Cyto-c was found to be 10 ng/mL and this new assay can be completed within 3h. Several potential anti-cancer drugs have been tested in vitro for their efficacy to kill liver cancer with or without multi-drug resistance. © 2013 Elsevier B.V. All rights reserved.

Magic Pools: Parallel Assessment of Transposon Delivery Vectors in Bacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Hualan; Price, Morgan N.; Waters, Robert Jordan

Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach for discovering the functions of bacterial genes. However, the development of a suitable TnSeq strategy for a given bacterium can be costly and time-consuming. To meet this challenge, we describe a part-based strategy for constructing libraries of hundreds of transposon delivery vectors, which we term “magic pools.” Within a magic pool, each transposon vector has a different combination of upstream sequences (promoters and ribosome binding sites) and antibiotic resistance markers as well as a random DNA barcode sequence, which allows the tracking of each vector during mutagenesis experiments. Tomore » identify an efficient vector for a given bacterium, we mutagenize it with a magic pool and sequence the resulting insertions; we then use this efficient vector to generate a large mutant library. We used the magic pool strategy to construct transposon mutant libraries in five genera of bacteria, including three genera of the phylumBacteroidetes. IMPORTANCEMolecular genetics is indispensable for interrogating the physiology of bacteria. However, the development of a functional genetic system for any given bacterium can be time-consuming. Here, we present a streamlined approach for identifying an effective transposon mutagenesis system for a new bacterium. Our strategy first involves the construction of hundreds of different transposon vector variants, which we term a “magic pool.” The efficacy of each vector in a magic pool is monitored in parallel using a unique DNA barcode that is introduced into each vector design. Using archived DNA “parts,” we next reassemble an effective vector for making a whole-genome transposon mutant library that is suitable for large-scale interrogation of gene function using competitive growth assays. Here, we demonstrate the utility of the magic pool system to make mutant libraries in five genera of bacteria.« less

Magic Pools: Parallel Assessment of Transposon Delivery Vectors in Bacteria

DOE PAGES

Liu, Hualan; Price, Morgan N.; Waters, Robert Jordan; ...

2018-01-16

Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach for discovering the functions of bacterial genes. However, the development of a suitable TnSeq strategy for a given bacterium can be costly and time-consuming. To meet this challenge, we describe a part-based strategy for constructing libraries of hundreds of transposon delivery vectors, which we term “magic pools.” Within a magic pool, each transposon vector has a different combination of upstream sequences (promoters and ribosome binding sites) and antibiotic resistance markers as well as a random DNA barcode sequence, which allows the tracking of each vector during mutagenesis experiments. Tomore » identify an efficient vector for a given bacterium, we mutagenize it with a magic pool and sequence the resulting insertions; we then use this efficient vector to generate a large mutant library. We used the magic pool strategy to construct transposon mutant libraries in five genera of bacteria, including three genera of the phylumBacteroidetes. IMPORTANCEMolecular genetics is indispensable for interrogating the physiology of bacteria. However, the development of a functional genetic system for any given bacterium can be time-consuming. Here, we present a streamlined approach for identifying an effective transposon mutagenesis system for a new bacterium. Our strategy first involves the construction of hundreds of different transposon vector variants, which we term a “magic pool.” The efficacy of each vector in a magic pool is monitored in parallel using a unique DNA barcode that is introduced into each vector design. Using archived DNA “parts,” we next reassemble an effective vector for making a whole-genome transposon mutant library that is suitable for large-scale interrogation of gene function using competitive growth assays. Here, we demonstrate the utility of the magic pool system to make mutant libraries in five genera of bacteria.« less

Environmental Mapping by a HERO-1 Robot Using Sonar and a Laser Barcode Scanner.

DTIC Science & Technology

1983-12-01

can be labled with an x-y type coordinate grid allowing the rover to directly read * its location as it moves along. A different approach is to...uses a two-dimensional grid of two character barcodes as reference objects. Since bar codes are designed to be read in either of two orientations (top...Processing Laboratory at AFIT (see Appendix B for listing). Navigation grid codes consist of two digits running consecutively from 00 to FF, yielding 256

A new universal, standardized implant database for product identification: a unique tool for arthroplasty registries.

PubMed

Blömer, Wilhelm; Steinbrück, Arnd; Schröder, Christian; Grothaus, Franz-Josef; Melsheimer, Oliver; Mannel, Henrich; Forkel, Gerhard; Eilers, Thomas; Liebs, Thoralf R; Hassenpflug, Joachim; Jansson, Volkmar

2015-07-01

Every joint registry aims to improve patient care by identifying implants that have an inferior performance. For this reason, each registry records the implant name that has been used in the individual patient. In most registries, a paper-based approach has been utilized for this purpose. However, in addition to being time-consuming, this approach does not account for the fact that failure patterns are not necessarily implant specific but can be associated with design features that are used in a number of implants. Therefore, we aimed to develop and evaluate an implant product library that allows both time saving barcode scanning on site in the hospital for the registration of the implant components and a detailed description of implant specifications. A task force consisting of representatives of the German Arthroplasty Registry, industry, and computer specialists agreed on a solution that allows barcode scanning of implant components and that also uses a detailed standardized classification describing arthroplasty components. The manufacturers classified all their components that are sold in Germany according to this classification. The implant database was analyzed regarding the completeness of components by algorithms and real-time data. The implant library could be set up successfully. At this point, the implant database includes more than 38,000 items, of which all were classified by the manufacturers according to the predefined scheme. Using patient data from the German Arthroplasty Registry, several errors in the database were detected, all of which were corrected by the respective implant manufacturers. The implant library that was developed for the German Arthroplasty Registry allows not only on-site barcode scanning for the registration of the implant components but also its classification tree allows a sophisticated analysis regarding implant characteristics, regardless of brand or manufacturer. The database is maintained by the implant manufacturers, thereby allowing registries to focus their resources on other areas of research. The database might represent a possible global model, which might encourage harmonization between joint replacement registries enabling comparisons between joint replacement registries.

Library preparation and data analysis packages for rapid genome sequencing.

PubMed

Pomraning, Kyle R; Smith, Kristina M; Bredeweg, Erin L; Connolly, Lanelle R; Phatale, Pallavi A; Freitag, Michael

2012-01-01

High-throughput sequencing (HTS) has quickly become a valuable tool for comparative genetics and genomics and is now regularly carried out in laboratories that are not connected to large sequencing centers. Here we describe an updated version of our protocol for constructing single- and paired-end Illumina sequencing libraries, beginning with purified genomic DNA. The present protocol can also be used for "multiplexing," i.e. the analysis of several samples in a single flowcell lane by generating "barcoded" or "indexed" Illumina sequencing libraries in a way that is independent from Illumina-supported methods. To analyze sequencing results, we suggest several independent approaches but end users should be aware that this is a quickly evolving field and that currently many alignment (or "mapping") and counting algorithms are being developed and tested.

Metabarcoding Is Powerful yet Still Blind: A Comparative Analysis of Morphological and Molecular Surveys of Seagrass Communities

PubMed Central

Cowart, Dominique A.; Pinheiro, Miguel; Mouchel, Olivier; Maguer, Marion; Grall, Jacques; Miné, Jacques; Arnaud-Haond, Sophie

2015-01-01

In the context of the sixth wave of extinction, reliable surveys of biodiversity are increasingly needed to infer the cause and consequences of species and community declines, identify early warning indicators of tipping points, and provide reliable impact assessments before engaging in activities with potential environmental hazards. DNA metabarcoding has emerged as having potential to provide speedy assessment of community structure from environmental samples. Here we tested the reliability of metabarcoding by comparing morphological and molecular inventories of invertebrate communities associated with seagrasses through estimates of alpha and beta diversity, as well as the identification of the most abundant taxa. Sediment samples were collected from six Zostera marina seagrass meadows across Brittany, France. Metabarcoding surveys were performed using both mitochondrial (Cytochrome Oxidase I) and nuclear (small subunit 18S ribosomal RNA) markers, and compared to morphological inventories compiled by a long-term benthic monitoring network. A sampling strategy was defined to enhance performance and accuracy of results by preventing the dominance of larger animals, boosting statistical support through replicates, and using two genes to compensate for taxonomic biases. Molecular barcodes proved powerful by revealing a remarkable level of diversity that vastly exceeded the morphological survey, while both surveys identified congruent differentiation of the meadows. However, despite the addition of individual barcodes of common species into taxonomic reference databases, the retrieval of only 36% of these species suggest that the remaining were either not present in the molecular samples or not detected by the molecular screening. This finding exemplifies the necessity of comprehensive and well-curated taxonomic reference libraries and multi-gene surveys. Overall, results offer methodological guidelines and support for metabarcoding as a powerful and repeatable method of characterizing communities, while also presenting suggestions for improvement, including implementation of pilot studies prior to performing full “blind” metabarcoding assessments to optimize sampling and amplification protocols. PMID:25668035

Development and application of a recombination-based library versus library high- throughput yeast two-hybrid (RLL-Y2H) screening system.

PubMed

Yang, Fang; Lei, Yingying; Zhou, Meiling; Yao, Qili; Han, Yichao; Wu, Xiang; Zhong, Wanshun; Zhu, Chenghang; Xu, Weize; Tao, Ran; Chen, Xi; Lin, Da; Rahman, Khaista; Tyagi, Rohit; Habib, Zeshan; Xiao, Shaobo; Wang, Dang; Yu, Yang; Chen, Huanchun; Fu, Zhenfang; Cao, Gang

2018-02-16

Protein-protein interaction (PPI) network maintains proper function of all organisms. Simple high-throughput technologies are desperately needed to delineate the landscape of PPI networks. While recent state-of-the-art yeast two-hybrid (Y2H) systems improved screening efficiency, either individual colony isolation, library preparation arrays, gene barcoding or massive sequencing are still required. Here, we developed a recombination-based 'library vs library' Y2H system (RLL-Y2H), by which multi-library screening can be accomplished in a single pool without any individual treatment. This system is based on the phiC31 integrase-mediated integration between bait and prey plasmids. The integrated fragments were digested by MmeI and subjected to deep sequencing to decode the interaction matrix. We applied this system to decipher the trans-kingdom interactome between Mycobacterium tuberculosis and host cells and further identified Rv2427c interfering with the phagosome-lysosome fusion. This concept can also be applied to other systems to screen protein-RNA and protein-DNA interactions and delineate signaling landscape in cells.

Pitfalls of Establishing DNA Barcoding Systems in Protists: The Cryptophyceae as a Test Case

PubMed Central

Hoef-Emden, Kerstin

2012-01-01

A DNA barcode is a preferrably short and highly variable region of DNA supposed to facilitate a rapid identification of species. In many protistan lineages, a lack of species-specific morphological characters hampers an identification of species by light or electron microscopy, and difficulties to perform mating experiments in laboratory cultures also do not allow for an identification of biological species. Thus, testing candidate barcode markers as well as establishment of accurately working species identification systems are more challenging than in multicellular organisms. In cryptic species complexes the performance of a potential barcode marker can not be monitored using morphological characters as a feedback, but an inappropriate choice of DNA region may result in artifactual species trees for several reasons. Therefore a priori knowledge of the systematics of a group is required. In addition to identification of known species, methods for an automatic delimitation of species with DNA barcodes have been proposed. The Cryptophyceae provide a mixture of systematically well characterized as well as badly characterized groups and are used in this study to test the suitability of some of the methods for protists. As species identification method the performance of blast in searches against badly to well-sampled reference databases has been tested with COI-5P and 5′-partial LSU rDNA (domains A to D of the nuclear LSU rRNA gene). In addition the performance of two different methods for automatic species delimitation, fixed thresholds of genetic divergence and the general mixed Yule-coalescent model (GMYC), have been examined. The study demonstrates some pitfalls of barcoding methods that have to be taken care of. Also a best-practice approach towards establishing a DNA barcode system in protists is proposed. PMID:22970104

Pitfalls of establishing DNA barcoding systems in protists: the cryptophyceae as a test case.

PubMed

Hoef-Emden, Kerstin

2012-01-01

A DNA barcode is a preferrably short and highly variable region of DNA supposed to facilitate a rapid identification of species. In many protistan lineages, a lack of species-specific morphological characters hampers an identification of species by light or electron microscopy, and difficulties to perform mating experiments in laboratory cultures also do not allow for an identification of biological species. Thus, testing candidate barcode markers as well as establishment of accurately working species identification systems are more challenging than in multicellular organisms. In cryptic species complexes the performance of a potential barcode marker can not be monitored using morphological characters as a feedback, but an inappropriate choice of DNA region may result in artifactual species trees for several reasons. Therefore a priori knowledge of the systematics of a group is required. In addition to identification of known species, methods for an automatic delimitation of species with DNA barcodes have been proposed. The Cryptophyceae provide a mixture of systematically well characterized as well as badly characterized groups and are used in this study to test the suitability of some of the methods for protists. As species identification method the performance of blast in searches against badly to well-sampled reference databases has been tested with COI-5P and 5'-partial LSU rDNA (domains A to D of the nuclear LSU rRNA gene). In addition the performance of two different methods for automatic species delimitation, fixed thresholds of genetic divergence and the general mixed Yule-coalescent model (GMYC), have been examined. The study demonstrates some pitfalls of barcoding methods that have to be taken care of. Also a best-practice approach towards establishing a DNA barcode system in protists is proposed.

Massively parallel cis-regulatory analysis in the mammalian central nervous system

PubMed Central

Shen, Susan Q.; Myers, Connie A.; Hughes, Andrew E.O.; Byrne, Leah C.; Flannery, John G.; Corbo, Joseph C.

2016-01-01

Cis-regulatory elements (CREs, e.g., promoters and enhancers) regulate gene expression, and variants within CREs can modulate disease risk. Next-generation sequencing has enabled the rapid generation of genomic data that predict the locations of CREs, but a bottleneck lies in functionally interpreting these data. To address this issue, massively parallel reporter assays (MPRAs) have emerged, in which barcoded reporter libraries are introduced into cells, and the resulting barcoded transcripts are quantified by next-generation sequencing. Thus far, MPRAs have been largely restricted to assaying short CREs in a limited repertoire of cultured cell types. Here, we present two advances that extend the biological relevance and applicability of MPRAs. First, we adapt exome capture technology to instead capture candidate CREs, thereby tiling across the targeted regions and markedly increasing the length of CREs that can be readily assayed. Second, we package the library into adeno-associated virus (AAV), thereby allowing delivery to target organs in vivo. As a proof of concept, we introduce a capture library of about 46,000 constructs, corresponding to roughly 3500 DNase I hypersensitive (DHS) sites, into the mouse retina by ex vivo plasmid electroporation and into the mouse cerebral cortex by in vivo AAV injection. We demonstrate tissue-specific cis-regulatory activity of DHSs and provide examples of high-resolution truncation mutation analysis for multiplex parsing of CREs. Our approach should enable massively parallel functional analysis of a wide range of CREs in any organ or species that can be infected by AAV, such as nonhuman primates and human stem cell–derived organoids. PMID:26576614

Integrative taxonomy detects cryptic and overlooked fish species in a neotropical river basin.

PubMed

Gomes, Laís Carvalho; Pessali, Tiago Casarim; Sales, Naiara Guimarães; Pompeu, Paulo Santos; Carvalho, Daniel Cardoso

2015-10-01

The great freshwater fish diversity found in the neotropical region makes management and conservation actions challenging. Due to shortage of taxonomists and insufficient infrastructure to deal with such great biodiversity (i.e. taxonomic impediment), proposed remedies to accelerate species identification and descriptions include techniques that combine DNA-based identification and concise morphological description. The building of a DNA barcode reference database correlating meristic and genetic data was developed for 75 % of the Mucuri River basin's freshwater fish. We obtained a total of 141 DNA barcode sequences from 37 species belonging to 30 genera, 19 families, and 5 orders. Genetic distances within species, genera, and families were 0.74, 9.5, and 18.86 %, respectively. All species could be clearly identified by the DNA barcodes. Divergences between meristic morphological characteristics and DNA barcodes revealed two cryptic species among the Cyphocharax gilbert and Astyanax gr. bimaculatus specimens, and helped to identify two overlooked species within the Gymnotus and Astyanax taxa. Therefore, using a simplified model of neotropical biodiversity, we tested the efficiency of an integrative taxonomy approach for species discovery, identification of cryptic diversity, and accelerating biodiversity descriptions.

Identification of Rays through DNA Barcoding: An Application for Ecologists

PubMed Central

Cerutti-Pereyra, Florencia; Meekan, Mark G.; Wei, Nu-Wei V.; O'Shea, Owen; Bradshaw, Corey J. A.; Austin, Chris M.

2012-01-01

DNA barcoding potentially offers scientists who are not expert taxonomists a powerful tool to support the accuracy of field studies involving taxa that are diverse and difficult to identify. The taxonomy of rays has received reasonable attention in Australia, although the fauna in remote locations such as Ningaloo Reef, Western Australia is poorly studied and the identification of some species in the field is problematic. Here, we report an application of DNA-barcoding to the identification of 16 species (from 10 genera) of tropical rays as part of an ecological study. Analysis of the dataset combined across all samples grouped sequences into clearly defined operational taxonomic units, with two conspicuous exceptions: the Neotrygon kuhlii species complex and the Aetobatus species complex. In the field, the group that presented the most difficulties for identification was the spotted whiptail rays, referred to as the ‘uarnak’ complex. Two sets of problems limited the successful application of DNA barcoding: (1) the presence of cryptic species, species complexes with unresolved taxonomic status and intra-specific geographical variation, and (2) insufficient numbers of entries in online databases that have been verified taxonomically, and the presence of lodged sequences in databases with inconsistent names. Nevertheless, we demonstrate the potential of the DNA barcoding approach to confirm field identifications and to highlight species complexes where taxonomic uncertainty might confound ecological data. PMID:22701556

DNA Barcoding of the Korean Lymantria Hübner, 1819 (Lepidoptera: Erebidae: Lymantriinae) for Quarantine Inspection.

PubMed

Kang, Tae Hwa; Lee, Kwang-Su; Lee, Heung-Sik

2015-08-01

DNA barcoding and morphological analyses of Korean Lymantria (Erebidae, Lepidoptera) were conducted for quarantine inspection. In DNA barcoding, Lymantria dispar identified through quarantine inspection was distinguished as three species, L. dispar asiatica, L. albescens, and L. xylina. Lymantria monacha, which is known as a single species in Korea, is revealed as containing three species, L. monacha, L. minomonis, and L. sugii. At the subspecies level, L. dispar dispar formed a single cluster, whereas L. d. asiatica and L. d. japonica formed a cluster containing both subspecies. In morphological re-examination on DNA barcoding results, L. dispar was distinguished from L. albescens by wing pattern, and from L. xylina by papillae anale. L. monacha and the related species were hard to be distinct from each other by using wing pattern, but it was easily distinct through comparison of genitalia. Therefore, DNA barcoding led to accurate identification in species level, but in subspecies level, only a taxon showing geographically far distance was discriminated from the others. These results may provide a taxonomic outline of the Korean Lymantria fauna and may be used as an identification reference for Lymantria species during quarantine inspection. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

TaxI: a software tool for DNA barcoding using distance methods

PubMed Central

Steinke, Dirk; Vences, Miguel; Salzburger, Walter; Meyer, Axel

2005-01-01

DNA barcoding is a promising approach to the diagnosis of biological diversity in which DNA sequences serve as the primary key for information retrieval. Most existing software for evolutionary analysis of DNA sequences was designed for phylogenetic analyses and, hence, those algorithms do not offer appropriate solutions for the rapid, but precise analyses needed for DNA barcoding, and are also unable to process the often large comparative datasets. We developed a flexible software tool for DNA taxonomy, named TaxI. This program calculates sequence divergences between a query sequence (taxon to be barcoded) and each sequence of a dataset of reference sequences defined by the user. Because the analysis is based on separate pairwise alignments this software is also able to work with sequences characterized by multiple insertions and deletions that are difficult to align in large sequence sets (i.e. thousands of sequences) by multiple alignment algorithms because of computational restrictions. Here, we demonstrate the utility of this approach with two datasets of fish larvae and juveniles from Lake Constance and juvenile land snails under different models of sequence evolution. Sets of ribosomal 16S rRNA sequences, characterized by multiple indels, performed as good as or better than cox1 sequence sets in assigning sequences to species, demonstrating the suitability of rRNA genes for DNA barcoding. PMID:16214755

DNA barcode reference data for the Korean herpetofauna and their applications.

PubMed

Jeong, Tae Jin; Jun, Jumin; Han, Sanghoon; Kim, Hyun Tae; Oh, Kyunghee; Kwak, Myounghai

2013-11-01

Recently, amphibians and reptiles have drawn attention because of declines in species and populations caused mainly by habitat loss, overexploitation and climate change. This study constructed a DNA barcode database for the Korean herpetofauna, including all the recorded amphibians and 68% of the recorded reptiles, to provide a useful, standardized tool for species identification in monitoring and management. A total of 103 individuals from 18 amphibian and 17 reptile species were used to generate barcode sequences using partial sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene and to compare it with other suggested barcode loci. Comparing 16S rRNA, cytochrome b (Cytb) and COI for amphibians and 12S rRNA, Cytb and COI for reptiles, our results revealed that COI is better than the other markers in terms of a high level of sequence variation without length variation and moderate amplification success. Although the COI marker had no clear barcoding gap because of the high level of intraspecific variation, all of the analysed individuals from the same species clustered together in a neighbour-joining tree. High intraspecific variation suggests the possibility of cryptic species. Finally, using this database, confiscated snakes were identified as Elaphe schrenckii, designated as endangered in Korea and a food contaminant was identified as the lizard Takydromus amurensis. © 2013 John Wiley & Sons Ltd.

Diversity of planktonic fish larvae along a latitudinal gradient in the Eastern Atlantic Ocean estimated through DNA barcodes

PubMed Central

Morote, Elvira; Kochzius, Marc; Garcia-Vazquez, Eva

2016-01-01

Mid-trophic pelagic fish are essential components of marine ecosystems because they represent the link between plankton and higher predators. Moreover, they are the basis of the most important fisheries resources; for example, in African waters. In this study, we have sampled pelagic fish larvae in the Eastern Atlantic Ocean along a latitudinal gradient between 37°N and 2°S. We have employed Bongo nets for plankton sampling and sorted visually fish and fish larvae. Using the cytochrome oxidase I gene (COI) as a DNA barcode, we have identified 44 OTUs down to species level that correspond to 14 families, with Myctophidae being the most abundant. A few species were cosmopolitan and others latitude-specific, as was expected. The latitudinal pattern of diversity did not exhibit a temperate-tropical cline; instead, it was likely correlated with environmental conditions with a decline in low-oxygen zones. Importantly, gaps and inconsistencies in reference DNA databases impeded accurate identification to the species level of 49% of the individuals. Fish sampled from tropical latitudes and some orders, such as Perciformes, Myctophiformes and Stomiiformes, were largely unidentified due to incomplete references. Some larvae were identified based on morphology and COI analysis for comparing time and costs employed from each methodology. These results suggest the need of reinforcing DNA barcoding reference datasets of Atlantic bathypelagic tropical fish that, as main prey of top predators, are crucial for ecosystem-based management of fisheries resources. PMID:27761307

Tropical montane nymphalids in Mexico: DNA barcodes reveal greater diversity.

PubMed

Escalante, Patricia; Ibarra-Vazquez, Adolfo; Rosas-Escobar, Patricia

2010-12-01

DNA sequences obtained for the Barcode of Life library in the All Lepidoptera Campaign project Nymphalidae of Central Mexico were analyzed as a test of species limits and to explore possible phylogenetic groupings in the Preponini tribe. Using specimens in the National Insect Collection of the Instituto de Biología of the Universidad Nacional Autónoma de México, 78 specimens were assayed for cytochrome oxidase c subunit 1. Disregarding the missing data, there were 458 conserved sites, 200 variable sites and 187 parsimony-informative sites. The neighbor-joining and maximum likelihood analyses indicate that none of the three genera of Preponini as currently circumscribed are reciprocally monophyletic. As per species limits, high levels of barcode variation in the Prepona deiphile complex suggest the existence of at least two new endemic species to Mexico. The divergent taxa were escalantiana from the Tuxtlas region in Veracruz, and ibarra from Sierra Madre del Sur in the Pacific states of southern Mexico. The genetic distance in the CO1 fragment between them and the other deiphile populations ranged from 2.7 to 8.0%. We recommend that morphological data need to be re-examined and that additional molecular data for species ought to be gathered before a particular biogeographic model can be proposed for the group in Mesoamerica.

DNA barcoding of human-biting black flies (Diptera: Simuliidae) in Thailand.

PubMed

Pramual, Pairot; Thaijarern, Jiraporn; Wongpakam, Komgrit

2016-12-01

Black flies (Diptera: Simuliidae) are important insect vectors and pests of humans and animals. Accurate identification, therefore, is important for control and management. In this study, we used mitochondrial cytochrome oxidase I (COI) barcoding sequences to test the efficiency of species identification for the human-biting black flies in Thailand. We used human-biting specimens because they enabled us to link information with previous studies involving the immature stages. Three black fly taxa, Simulium nodosum, S. nigrogilvum and S. doipuiense complex, were collected. The S. doipuiense complex was confirmed for the first time as having human-biting habits. The COI sequences revealed considerable genetic diversity in all three species. Comparisons to a COI sequence library of black flies in Thailand and in a public database indicated a high efficiency for specimen identification for S. nodosum and S. nigrogilvum, but this method was not successful for the S. doipuiense complex. Phylogenetic analyses revealed two divergent lineages in the S. doipuiense complex. Human-biting specimens formed a separate clade from other members of this complex. The results are consistent with the Barcoding Index Number System (BINs) analysis that found six BINs in the S. doipuiense complex. Further taxonomic work is needed to clarify the species status of these human-biting specimens. Copyright © 2016 Elsevier B.V. All rights reserved.

Functional annotation of chemical libraries across diverse biological processes.

PubMed

Piotrowski, Jeff S; Li, Sheena C; Deshpande, Raamesh; Simpkins, Scott W; Nelson, Justin; Yashiroda, Yoko; Barber, Jacqueline M; Safizadeh, Hamid; Wilson, Erin; Okada, Hiroki; Gebre, Abraham A; Kubo, Karen; Torres, Nikko P; LeBlanc, Marissa A; Andrusiak, Kerry; Okamoto, Reika; Yoshimura, Mami; DeRango-Adem, Eva; van Leeuwen, Jolanda; Shirahige, Katsuhiko; Baryshnikova, Anastasia; Brown, Grant W; Hirano, Hiroyuki; Costanzo, Michael; Andrews, Brenda; Ohya, Yoshikazu; Osada, Hiroyuki; Yoshida, Minoru; Myers, Chad L; Boone, Charles

2017-09-01

Chemical-genetic approaches offer the potential for unbiased functional annotation of chemical libraries. Mutations can alter the response of cells in the presence of a compound, revealing chemical-genetic interactions that can elucidate a compound's mode of action. We developed a highly parallel, unbiased yeast chemical-genetic screening system involving three key components. First, in a drug-sensitive genetic background, we constructed an optimized diagnostic mutant collection that is predictive for all major yeast biological processes. Second, we implemented a multiplexed (768-plex) barcode-sequencing protocol, enabling the assembly of thousands of chemical-genetic profiles. Finally, based on comparison of the chemical-genetic profiles with a compendium of genome-wide genetic interaction profiles, we predicted compound functionality. Applying this high-throughput approach, we screened seven different compound libraries and annotated their functional diversity. We further validated biological process predictions, prioritized a diverse set of compounds, and identified compounds that appear to have dual modes of action.

Chat reference service in medical libraries: part 2--Trends in medical school libraries.

PubMed

Dee, Cheryl R

2003-01-01

An increasing number of medical school libraries offer chat service to provide immediate, high quality information at the time and point of need to students, faculty, staff, and health care professionals. Part 2 of Chat Reference Service in Medical Libraries presents a snapshot of the current trends in chat reference service in medical school libraries. In late 2002, 25 (21%) medical school libraries provided chat reference. Trends in chat reference services in medical school libraries were compiled from an exploration of medical school library Web sites and informal correspondence from medical school library personnel. Many medical libraries are actively investigating and planning new chat reference services, while others have decided not to pursue chat reference at this time. Anecdotal comments from medical school library staff provide insights into chat reference service.

Deep COI sequencing of standardized benthic samples unveils overlooked diversity of Jordanian coral reefs in the northern Red Sea.

PubMed

Al-Rshaidat, Mamoon M D; Snider, Allison; Rosebraugh, Sydney; Devine, Amanda M; Devine, Thomas D; Plaisance, Laetitia; Knowlton, Nancy; Leray, Matthieu

2016-09-01

High-throughput sequencing (HTS) of DNA barcodes (metabarcoding), particularly when combined with standardized sampling protocols, is one of the most promising approaches for censusing overlooked cryptic invertebrate communities. We present biodiversity estimates based on sequencing of the cytochrome c oxidase subunit 1 (COI) gene for coral reefs of the Gulf of Aqaba, a semi-enclosed system in the northern Red Sea. Samples were obtained from standardized sampling devices (Autonomous Reef Monitoring Structures (ARMS)) deployed for 18 months. DNA barcoding of non-sessile specimens >2 mm revealed 83 OTUs in six phyla, of which only 25% matched a reference sequence in public databases. Metabarcoding of the 2 mm - 500 μm and sessile bulk fractions revealed 1197 OTUs in 15 animal phyla, of which only 4.9% matched reference barcodes. These results highlight the scarcity of COI data for cryptobenthic organisms of the Red Sea. Compared with data obtained using similar methods, our results suggest that Gulf of Aqaba reefs are less diverse than two Pacific coral reefs but much more diverse than an Atlantic oyster reef at a similar latitude. The standardized approaches used here show promise for establishing baseline data on biodiversity, monitoring the impacts of environmental change, and quantifying patterns of diversity at regional and global scales.

DNA barcoding and traditional taxonomy: an integrated approach for biodiversity conservation.

PubMed

Sheth, Bhavisha P; Thaker, Vrinda S

2017-07-01

Biological diversity is depleting at an alarming rate. Additionally, a vast amount of biodiversity still remains undiscovered. Taxonomy has been serving the purpose of describing, naming, and classifying species for more than 250 years. DNA taxonomy and barcoding have accelerated the rate of this process, thereby providing a tool for conservation practice. DNA barcoding and traditional taxonomy have their own inherent merits and demerits. The synergistic use of both methods, in the form of integrative taxonomy, has the potential to contribute to biodiversity conservation in a pragmatic timeframe and overcome their individual drawbacks. In this review, we discuss the basics of both these methods of biological identification (traditional taxonomy and DNA barcoding), the technical advances in integrative taxonomy, and future trends. We also present a comprehensive compilation of published examples of integrative taxonomy that refer to nine topics within biodiversity conservation. Morphological and molecular species limits were observed to be congruent in ∼41% of the 58 source studies. The majority of the studies highlighted the description of cryptic diversity through the use of molecular data, whereas research areas like endemism, biological invasion, and threatened species were less discussed in the literature.

The problems and promise of DNA barcodes for species diagnosis of primate biomaterials

PubMed Central

Lorenz, Joseph G; Jackson, Whitney E; Beck, Jeanne C; Hanner, Robert

2005-01-01

The Integrated Primate Biomaterials and Information Resource (www.IPBIR.org) provides essential research reagents to the scientific community by establishing, verifying, maintaining, and distributing DNA and RNA derived from primate cell cultures. The IPBIR uses mitochondrial cytochrome c oxidase subunit I sequences to verify the identity of samples for quality control purposes in the accession, cell culture, DNA extraction processes and prior to shipping to end users. As a result, IPBIR is accumulating a database of ‘DNA barcodes’ for many species of primates. However, this quality control process is complicated by taxon specific patterns of ‘universal primer’ failure, as well as the amplification or co-amplification of nuclear pseudogenes of mitochondrial origins. To overcome these difficulties, taxon specific primers have been developed, and reverse transcriptase PCR is utilized to exclude these extraneous sequences from amplification. DNA barcoding of primates has applications to conservation and law enforcement. Depositing barcode sequences in a public database, along with primer sequences, trace files and associated quality scores, makes this species identification technique widely accessible. Reference DNA barcode sequences should be derived from, and linked to, specimens of known provenance in web-accessible collections in order to validate this system of molecular diagnostics. PMID:16214744

Molecular species identification with rich floristic sampling: DNA barcoding the pteridophyte flora of Japan.

PubMed

Ebihara, Atsushi; Nitta, Joel H; Ito, Motomi

2010-12-08

DNA barcoding is expected to be an effective identification tool for organisms with heteromorphic generations such as pteridophytes, which possess a morphologically simple gametophyte generation. Although a reference data set including complete coverage of the target local flora/fauna is necessary for accurate identification, DNA barcode studies including such rich taxonomic sampling on a countrywide scale are lacking. The Japanese pteridophyte flora (733 taxa including subspecies and varieties) was used to test the utility of two plastid DNA barcode regions (rbcL and trnH-psbA) with the intention of developing an identification system for native gametophytes. DNA sequences were obtained from each of 689 (94.0%) taxa for rbcL and 617 (84.2%) taxa for trnH-psbA. Mean interspecific divergence values across all taxon pairs (K2P genetic distances) did not reveal a significant difference in rate between trnH-psbA and rbcL, but mean K2P distances of each genus showed significant heterogeneity according to systematic position. The minimum fail rate of taxon discrimination in an identification test using BLAST (12.52%) was obtained when rbcL and trnH-psbA were combined, and became lower in datasets excluding infraspecific taxa or apogamous taxa, or including sexual diploids only. This study demonstrates the overall effectiveness of DNA barcodes for species identification in the Japanese pteridophyte flora. Although this flora is characterized by a high occurrence of apogamous taxa that pose a serious challenge to identification using DNA barcodes, such taxa are limited to a small number of genera, and only minimally detract from the overall success rate. In the case that a query sequence is matched to a known apogamous genus, routine species identification may not be possible. Otherwise, DNA barcoding is a practical tool for identification of most Japanese pteridophytes, and is especially anticipated to be helpful for identification of non-hybridizing gametophytes.

Genome-Wide Tuning of Protein Expression Levels to Rapidly Engineer Microbial Traits.

PubMed

Freed, Emily F; Winkler, James D; Weiss, Sophie J; Garst, Andrew D; Mutalik, Vivek K; Arkin, Adam P; Knight, Rob; Gill, Ryan T

2015-11-20

The reliable engineering of biological systems requires quantitative mapping of predictable and context-independent expression over a broad range of protein expression levels. However, current techniques for modifying expression levels are cumbersome and are not amenable to high-throughput approaches. Here we present major improvements to current techniques through the design and construction of E. coli genome-wide libraries using synthetic DNA cassettes that can tune expression over a ∼10(4) range. The cassettes also contain molecular barcodes that are optimized for next-generation sequencing, enabling rapid and quantitative tracking of alleles that have the highest fitness advantage. We show these libraries can be used to determine which genes and expression levels confer greater fitness to E. coli under different growth conditions.

Assessment, validation and deployment strategy of a two-barcode protocol for facile genotyping of duckweed species.

PubMed

Borisjuk, N; Chu, P; Gutierrez, R; Zhang, H; Acosta, K; Friesen, N; Sree, K S; Garcia, C; Appenroth, K J; Lam, E

2015-01-01

Lemnaceae, commonly called duckweeds, comprise a diverse group of floating aquatic plants that have previously been classified into 37 species based on morphological and physiological criteria. In addition to their unique evolutionary position among angiosperms and their applications in biomonitoring, the potential of duckweeds as a novel sustainable crop for fuel and feed has recently increased interest in the study of their biodiversity and systematics. However, due to their small size and abbreviated structure, accurate typing of duckweeds based on morphology can be challenging. In the past decade, attempts to employ molecular barcoding techniques for species assignment have produced promising results; however, they have yet to be codified into a simple and quantitative protocol. A study that compiles and compares the barcode sequences within all known species of this family would help to establish the fidelity and limits of this DNA-based approach. In this work, we compared the level of conservation between over 100 strains of duckweed for two intergenic barcode sequences derived from the plastid genome. By using over 300 sequences publicly available in the NCBI database, we determined the utility of each of these two barcodes for duckweed species identification. Through sequencing of these barcodes from additional accessions, 30 of the 37 known species of duckweed could be identified with varying levels of confidence using this approach. From our analyses using this reference dataset, we also confirmed two instances where mis-assignment of species has likely occurred. Potential strategies for further improving the scope of this technology are discussed. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

DNA barcoding for the identification of sand fly species (Diptera, Psychodidae, Phlebotominae) in Colombia.

PubMed

Contreras Gutiérrez, María Angélica; Vivero, Rafael J; Vélez, Iván D; Porter, Charles H; Uribe, Sandra

2014-01-01

Sand flies include a group of insects that are of medical importance and that vary in geographic distribution, ecology, and pathogen transmission. Approximately 163 species of sand flies have been reported in Colombia. Surveillance of the presence of sand fly species and the actualization of species distribution are important for predicting risks for and monitoring the expansion of diseases which sand flies can transmit. Currently, the identification of phlebotomine sand flies is based on morphological characters. However, morphological identification requires considerable skills and taxonomic expertise. In addition, significant morphological similarity between some species, especially among females, may cause difficulties during the identification process. DNA-based approaches have become increasingly useful and promising tools for estimating sand fly diversity and for ensuring the rapid and accurate identification of species. A partial sequence of the mitochondrial cytochrome oxidase gene subunit I (COI) is currently being used to differentiate species in different animal taxa, including insects, and it is referred as a barcoding sequence. The present study explored the utility of the DNA barcode approach for the identification of phlebotomine sand flies in Colombia. We sequenced 700 bp of the COI gene from 36 species collected from different geographic localities. The COI barcode sequence divergence within a single species was <2% in most cases, whereas this divergence ranged from 9% to 26.6% among different species. These results indicated that the barcoding gene correctly discriminated among the previously morphologically identified species with an efficacy of nearly 100%. Analyses of the generated sequences indicated that the observed species groupings were consistent with the morphological identifications. In conclusion, the barcoding gene was useful for species discrimination in sand flies from Colombia.

DNA Barcoding for the Identification of Sand Fly Species (Diptera, Psychodidae, Phlebotominae) in Colombia

PubMed Central

Contreras Gutiérrez, María Angélica; Vivero, Rafael J.; Vélez, Iván D.; Porter, Charles H.; Uribe, Sandra

2014-01-01

Sand flies include a group of insects that are of medical importance and that vary in geographic distribution, ecology, and pathogen transmission. Approximately 163 species of sand flies have been reported in Colombia. Surveillance of the presence of sand fly species and the actualization of species distribution are important for predicting risks for and monitoring the expansion of diseases which sand flies can transmit. Currently, the identification of phlebotomine sand flies is based on morphological characters. However, morphological identification requires considerable skills and taxonomic expertise. In addition, significant morphological similarity between some species, especially among females, may cause difficulties during the identification process. DNA-based approaches have become increasingly useful and promising tools for estimating sand fly diversity and for ensuring the rapid and accurate identification of species. A partial sequence of the mitochondrial cytochrome oxidase gene subunit I (COI) is currently being used to differentiate species in different animal taxa, including insects, and it is referred as a barcoding sequence. The present study explored the utility of the DNA barcode approach for the identification of phlebotomine sand flies in Colombia. We sequenced 700 bp of the COI gene from 36 species collected from different geographic localities. The COI barcode sequence divergence within a single species was <2% in most cases, whereas this divergence ranged from 9% to 26.6% among different species. These results indicated that the barcoding gene correctly discriminated among the previously morphologically identified species with an efficacy of nearly 100%. Analyses of the generated sequences indicated that the observed species groupings were consistent with the morphological identifications. In conclusion, the barcoding gene was useful for species discrimination in sand flies from Colombia. PMID:24454877

DNA Barcoding to Improve the Taxonomy of the Afrotropical Hoverflies (Insecta: Diptera: Syrphidae)

PubMed Central

Jordaens, Kurt; Goergen, Georg; Virgilio, Massimiliano; Backeljau, Thierry; Vokaer, Audrey; De Meyer, Marc

2015-01-01

The identification of Afrotropical hoverflies is very difficult because of limited recent taxonomic revisions and the lack of comprehensive identification keys. In order to assist in their identification, and to improve the taxonomy of this group, we constructed a reference dataset of 513 COI barcodes of 90 of the more common nominal species from Ghana, Togo, Benin and Nigeria (W Africa) and added ten publically available COI barcodes from nine nominal Afrotropical species to this (total: 523 COI barcodes; 98 nominal species; 26 genera). The identification accuracy of this dataset was evaluated with three methods (K2P distance-based, Neighbor-Joining (NJ) / Maximum Likelihood (ML) analysis, and using SpeciesIdentifier). Results of the three methods were highly congruent and showed a high identification success. Nine species pairs showed a low (< 0.03) mean interspecific K2P distance that resulted in several incorrect identifications. A high (> 0.03) maximum intraspecific K2P distance was observed in eight species and barcodes of these species not always formed single clusters in the NJ / ML analayses which may indicate the occurrence of cryptic species. Optimal K2P thresholds to differentiate intra- from interspecific K2P divergence were highly different among the three subfamilies (Eristalinae: 0.037, Syrphinae: 0.06, Microdontinae: 0.007–0.02), and among the different general suggesting that optimal thresholds are better defined at the genus level. In addition to providing an alternative identification tool, our study indicates that DNA barcoding improves the taxonomy of Afrotropical hoverflies by selecting (groups of) taxa that deserve further taxonomic study, and by attributing the unknown sex to species for which only one of the sexes is known. PMID:26473612

DNA Barcoding Green Microalgae Isolated from Neotropical Inland Waters

PubMed Central

Hadi, Sámed I. I. A.; Santana, Hugo; Brunale, Patrícia P. M.; Gomes, Taísa G.; Oliveira, Márcia D.; Matthiensen, Alexandre; Oliveira, Marcos E. C.; Silva, Flávia C. P.; Brasil, Bruno S. A. F.

2016-01-01

This study evaluated the feasibility of using the Ribulose Bisphosphate Carboxylase Large subunit gene (rbcL) and the Internal Transcribed Spacers 1 and 2 of the nuclear rDNA (nuITS1 and nuITS2) markers for identifying a very diverse, albeit poorly known group, of green microalgae from neotropical inland waters. Fifty-one freshwater green microalgae strains isolated from Brazil, the largest biodiversity reservoir in the neotropics, were submitted to DNA barcoding. Currently available universal primers for ITS1-5.8S-ITS2 region amplification were sufficient to successfully amplify and sequence 47 (92%) of the samples. On the other hand, new sets of primers had to be designed for rbcL, which allowed 96% of the samples to be sequenced. Thirty-five percent of the strains could be unambiguously identified to the species level based either on nuITS1 or nuITS2 sequences’ using barcode gap calculations. nuITS2 Compensatory Base Change (CBC) and ITS1-5.8S-ITS2 region phylogenetic analysis, together with morphological inspection, confirmed the identification accuracy. In contrast, only 6% of the strains could be assigned to the correct species based solely on rbcL sequences. In conclusion, the data presented here indicates that either nuITS1 or nuITS2 are useful markers for DNA barcoding of freshwater green microalgae, with advantage for nuITS2 due to the larger availability of analytical tools and reference barcodes deposited at databases for this marker. PMID:26900844

Quantitative Tracking of Combinatorially Engineered Populations with Multiplexed Binary Assemblies.

PubMed

Zeitoun, Ramsey I; Pines, Gur; Grau, Willliam C; Gill, Ryan T

2017-04-21

Advances in synthetic biology and genomics have enabled full-scale genome engineering efforts on laboratory time scales. However, the absence of sufficient approaches for mapping engineered genomes at system-wide scales onto performance has limited the adoption of more sophisticated algorithms for engineering complex biological systems. Here we report on the development and application of a robust approach to quantitatively map combinatorially engineered populations at scales up to several dozen target sites. This approach works by assembling genome engineered sites with cell-specific barcodes into a format compatible with high-throughput sequencing technologies. This approach, called barcoded-TRACE (bTRACE) was applied to assess E. coli populations engineered by recursive multiplex recombineering across both 6-target sites and 31-target sites. The 31-target library was then tracked throughout growth selections in the presence and absence of isopentenol (a potential next-generation biofuel). We also use the resolution of bTRACE to compare the influence of technical and biological noise on genome engineering efforts.

Polymers mediate a one-pot route for functionalized quantum dot barcodes with a large encoding capacity.

PubMed

Zhang, Ding Sheng-Zi; Jiang, Yang; Wei, Dan; Wei, Xunbin; Xu, Hong; Gu, Hongchen

2018-06-21

With the increasing demands for high-throughput multiplexed bioassays, quantum dot (QD)-encoded microbeads as biocarriers for various bioreactions have attracted considerable attention. However, three key requirements for these biocarriers are still longstanding issues: a stable fluorescence intensity, a large encoding capacity and abundant surface functional groups. Here, a novel one-pot strategy is developed, generating functionalized QD-encoded microspheres with a strong fluorescence intensity and optical stability. With poly(styrene-co-maleic anhydride) (PSMA) molecules as mediators, the encapsulation of QDs and carboxylation of the bead surface are integrated together, greatly improving the preparation efficiency and guaranteeing their potential application in biodetection. Moreover, the mechanism for preparing QD-doped beads is further proposed, which helps to precisely manipulate the preparation process and accurately encode the beads. Through this approach, a single- and dual-color barcode library of QD-encoded microspheres has been successfully established, which demonstrates their great potential in suspension arrays.

Construction and Analysis of Two Genome-Scale Deletion Libraries for Bacillus subtilis.

PubMed

Koo, Byoung-Mo; Kritikos, George; Farelli, Jeremiah D; Todor, Horia; Tong, Kenneth; Kimsey, Harvey; Wapinski, Ilan; Galardini, Marco; Cabal, Angelo; Peters, Jason M; Hachmann, Anna-Barbara; Rudner, David Z; Allen, Karen N; Typas, Athanasios; Gross, Carol A

2017-03-22

A systems-level understanding of Gram-positive bacteria is important from both an environmental and health perspective and is most easily obtained when high-quality, validated genomic resources are available. To this end, we constructed two ordered, barcoded, erythromycin-resistance- and kanamycin-resistance-marked single-gene deletion libraries of the Gram-positive model organism, Bacillus subtilis. The libraries comprise 3,968 and 3,970 genes, respectively, and overlap in all but four genes. Using these libraries, we update the set of essential genes known for this organism, provide a comprehensive compendium of B. subtilis auxotrophic genes, and identify genes required for utilizing specific carbon and nitrogen sources, as well as those required for growth at low temperature. We report the identification of enzymes catalyzing several missing steps in amino acid biosynthesis. Finally, we describe a suite of high-throughput phenotyping methodologies and apply them to provide a genome-wide analysis of competence and sporulation. Altogether, we provide versatile resources for studying gene function and pathway and network architecture in Gram-positive bacteria. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Advances in DNA metabarcoding for food and wildlife forensic species identification.

PubMed

Staats, Martijn; Arulandhu, Alfred J; Gravendeel, Barbara; Holst-Jensen, Arne; Scholtens, Ingrid; Peelen, Tamara; Prins, Theo W; Kok, Esther

2016-07-01

Species identification using DNA barcodes has been widely adopted by forensic scientists as an effective molecular tool for tracking adulterations in food and for analysing samples from alleged wildlife crime incidents. DNA barcoding is an approach that involves sequencing of short DNA sequences from standardized regions and comparison to a reference database as a molecular diagnostic tool in species identification. In recent years, remarkable progress has been made towards developing DNA metabarcoding strategies, which involves next-generation sequencing of DNA barcodes for the simultaneous detection of multiple species in complex samples. Metabarcoding strategies can be used in processed materials containing highly degraded DNA e.g. for the identification of endangered and hazardous species in traditional medicine. This review aims to provide insight into advances of plant and animal DNA barcoding and highlights current practices and recent developments for DNA metabarcoding of food and wildlife forensic samples from a practical point of view. Special emphasis is placed on new developments for identifying species listed in the Convention on International Trade of Endangered Species (CITES) appendices for which reliable methods for species identification may signal and/or prevent illegal trade. Current technological developments and challenges of DNA metabarcoding for forensic scientists will be assessed in the light of stakeholders' needs.

DNA Barcoding analysis of seafood accuracy in Washington, D.C. restaurants

PubMed Central

Stern, David B.; Castro Nallar, Eduardo; Rathod, Jason

2017-01-01

In Washington D.C., recent legislation authorizes citizens to test if products are properly represented and, if they are not, to bring a lawsuit for the benefit of the general public. Recent studies revealing the widespread phenomenon of seafood substitution across the United States make it a fertile area for consumer protection testing. DNA barcoding provides an accurate and cost-effective way to perform these tests, especially when tissue alone is available making species identification based on morphology impossible. In this study, we sequenced the 5′ barcoding region of the Cytochrome Oxidase I gene for 12 samples of vertebrate and invertebrate food items across six restaurants in Washington, D.C. and used multiple analytical methods to make identifications. These samples included several ambiguous menu listings, sequences with little genetic variation among closely related species and one sequence with no available reference sequence. Despite these challenges, we were able to make identifications for all samples and found that 33% were potentially mislabeled. While we found a high degree of mislabeling, the errors involved closely related species and we did not identify egregious substitutions as have been found in other cities. This study highlights the efficacy of DNA barcoding and robust analyses in identifying seafood items for consumer protection. PMID:28462038

Limited performance of DNA barcoding in a diverse community of tropical butterflies

PubMed Central

Elias, Marianne; Hill, Ryan I; Willmott, Keith R; Dasmahapatra, Kanchon K; Brower, Andrew V.Z; Mallet, James; Jiggins, Chris D

2007-01-01

DNA ‘barcoding’ relies on a short fragment of mitochondrial DNA to infer identification of specimens. The method depends on genetic diversity being markedly lower within than between species. Closely related species are most likely to share genetic variation in communities where speciation rates are rapid and effective population sizes are large, such that coalescence times are long. We assessed the applicability of DNA barcoding (here the 5′ half of the cytochrome c oxidase I) to a diverse community of butterflies from the upper Amazon, using a group with a well-established morphological taxonomy to serve as a reference. Only 77% of species could be accurately identified using the barcode data, a figure that dropped to 68% in species represented in the analyses by more than one geographical race and at least one congener. The use of additional mitochondrial sequence data hardly improved species identification, while a fragment of a nuclear gene resolved issues in some of the problematic species. We acknowledge the utility of barcodes when morphological characters are ambiguous or unknown, but we also recommend the addition of nuclear sequence data, and caution that species-level identification rates might be lower in the most diverse habitats of our planet. PMID:17785265

DNA barcode based wildlife forensics for resolving the origin of claw samples using a novel primer cocktail.

PubMed

Khedkar, Gulab D; Abhayankar, Shil Bapurao; Nalage, Dinesh; Ahmed, Shaikh Nadeem; Khedkar, Chandraprakash D

2016-11-01

Excessive wildlife hunting for commercial purposes can have negative impacts on biodiversity and may result in species extinction. To ensure compliance with legal statutes, forensic identification approaches relying on molecular markers may be used to identify the species of origin of animal material from hairs, claw, blood, bone, or meat. Using this approach, DNA sequences from the COI "barcoding" gene have been used to identify material from a number of domesticated animal species. However, many wild species of carnivores still present great challenges in generating COI barcodes using standard "universal" primer pairs. In the work presented here, the mitochondrial COI gene was successfully amplified using a novel primer cocktail, and the products were sequenced to determine the species of twenty one unknown samples of claw material collected as part of forensic wildlife case investigations. Sixteen of the unknown samples were recognized to have originated from either Panthera leo or P. pardus individuals. The remaining five samples could be identified only to the family level due to the absence of reference animal sequences. This is the first report on the use of COI sequences for the identification of P. pardus and P. leo from claw samples as part of forensic investigations in India. The study also highlights the need for adequate reference material to aid in the resolution of suspected cases of illegal wildlife harvesting.

DNA barcoding amphibians and reptiles.

PubMed

Vences, Miguel; Nagy, Zoltán T; Sonet, Gontran; Verheyen, Erik

2012-01-01

Only a few major research programs are currently targeting COI barcoding of amphibians and reptiles (including chelonians and crocodiles), two major groups of tetrapods. Amphibian and reptile species are typically old, strongly divergent, and contain deep conspecific lineages which might lead to problems in species assignment with incomplete reference databases. As far as known, there is no single pair of COI primers that will guarantee a sufficient rate of success across all amphibian and reptile taxa, or within major subclades of amphibians and reptiles, which means that the PCR amplification strategy needs to be adjusted depending on the specific research question. In general, many more amphibian and reptile taxa have been sequenced for 16S rDNA, which for some purposes may be a suitable complementary marker, at least until a more comprehensive COI reference database becomes available. DNA barcoding has successfully been used to identify amphibian larval stages (tadpoles) in species-rich tropical assemblages. Tissue sampling, DNA extraction, and amplification of COI is straightforward in amphibians and reptiles. Single primer pairs are likely to have a failure rate between 5 and 50% if taxa of a wide taxonomic range are targeted; in such cases the use of primer cocktails or subsequent hierarchical usage of different primer pairs is necessary. If the target group is taxonomically limited, many studies have followed a strategy of designing specific primers which then allow an easy and reliable amplification of all samples.

Highly multiplexed targeted DNA sequencing from single nuclei.

PubMed

Leung, Marco L; Wang, Yong; Kim, Charissa; Gao, Ruli; Jiang, Jerry; Sei, Emi; Navin, Nicholas E

2016-02-01

Single-cell DNA sequencing methods are challenged by poor physical coverage, high technical error rates and low throughput. To address these issues, we developed a single-cell DNA sequencing protocol that combines flow-sorting of single nuclei, time-limited multiple-displacement amplification (MDA), low-input library preparation, DNA barcoding, targeted capture and next-generation sequencing (NGS). This approach represents a major improvement over our previous single nucleus sequencing (SNS) Nature Protocols paper in terms of generating higher-coverage data (>90%), thereby enabling the detection of genome-wide variants in single mammalian cells at base-pair resolution. Furthermore, by pooling 48-96 single-cell libraries together for targeted capture, this approach can be used to sequence many single-cell libraries in parallel in a single reaction. This protocol greatly reduces the cost of single-cell DNA sequencing, and it can be completed in 5-6 d by advanced users. This single-cell DNA sequencing protocol has broad applications for studying rare cells and complex populations in diverse fields of biological research and medicine.

Using single nuclei for RNA-seq to capture the transcriptome of postmortem neurons

PubMed Central

Krishnaswami, Suguna Rani; Grindberg, Rashel V; Novotny, Mark; Venepally, Pratap; Lacar, Benjamin; Bhutani, Kunal; Linker, Sara B; Pham, Son; Erwin, Jennifer A; Miller, Jeremy A; Hodge, Rebecca; McCarthy, James K; Kelder, Martin; McCorrison, Jamison; Aevermann, Brian D; Fuertes, Francisco Diez; Scheuermann, Richard H; Lee, Jun; Lein, Ed S; Schork, Nicholas; McConnell, Michael J; Gage, Fred H; Lasken, Roger S

2016-01-01

A protocol is described for sequencing the transcriptome of a cell nucleus. Nuclei are isolated from specimens and sorted by FACS, cDNA libraries are constructed and RNA-seq is performed, followed by data analysis. Some steps follow published methods (Smart-seq2 for cDNA synthesis and Nextera XT barcoded library preparation) and are not described in detail here. Previous single-cell approaches for RNA-seq from tissues include cell dissociation using protease treatment at 30 °C, which is known to alter the transcriptome. We isolate nuclei at 4 °C from tissue homogenates, which cause minimal damage. Nuclear transcriptomes can be obtained from postmortem human brain tissue stored at −80 °C, making brain archives accessible for RNA-seq from individual neurons. The method also allows investigation of biological features unique to nuclei, such as enrichment of certain transcripts and precursors of some noncoding RNAs. By following this procedure, it takes about 4 d to construct cDNA libraries that are ready for sequencing. PMID:26890679

Digital reference service: trends in academic health science libraries.

PubMed

Dee, Cheryl R

2005-01-01

Two years after the initial 2002 study, a greater number of academic health science libraries are offering digital reference chat services, and this number appears poised to grow in the coming years. This 2004 follow-up study found that 36 (27%) of the academic health science libraries examined provide digital chat reference services; this was an approximately 6% increase over the 25 libraries (21%) located in 2002. Trends in digital reference services in academic health science libraries were derived from the exploration of academic health science library Web sites and from digital correspondence with academic health science library personnel using e-mail and chat. This article presents an overview of the current state of digital reference service in academic health science libraries.

Rethinking Virtual Reference

ERIC Educational Resources Information Center

Tenopir, Carol

2004-01-01

Virtual reference services seem a natural extension of libraries digital collections and the emphasis on access to the library anytime, anywhere. If patrons use the library from home, it makes sense to provide them with person-to-person online reference. The Library of Congress (LC), OCLC, and several large library systems have developed and…

Genomic phenotyping by barcode sequencing broadly distinguishes between alkylating agents, oxidizing agents, and non-genotoxic agents, and reveals a role for aromatic amino acids in cellular recovery after quinone exposure.

PubMed

Svensson, J Peter; Quirós Pesudo, Laia; McRee, Siobhan K; Adeleye, Yeyejide; Carmichael, Paul; Samson, Leona D

2013-01-01

Toxicity screening of compounds provides a means to identify compounds harmful for human health and the environment. Here, we further develop the technique of genomic phenotyping to improve throughput while maintaining specificity. We exposed cells to eight different compounds that rely on different modes of action: four genotoxic alkylating (methyl methanesulfonate (MMS), N-Methyl-N-nitrosourea (MNU), N,N'-bis(2-chloroethyl)-N-nitroso-urea (BCNU), N-ethylnitrosourea (ENU)), two oxidizing (2-methylnaphthalene-1,4-dione (menadione, MEN), benzene-1,4-diol (hydroquinone, HYQ)), and two non-genotoxic (methyl carbamate (MC) and dimethyl sulfoxide (DMSO)) compounds. A library of S. cerevisiae 4,852 deletion strains, each identifiable by a unique genetic 'barcode', were grown in competition; at different time points the ratio between the strains was assessed by quantitative high throughput 'barcode' sequencing. The method was validated by comparison to previous genomic phenotyping studies and 90% of the strains identified as MMS-sensitive here were also identified as MMS-sensitive in a much lower throughput solid agar screen. The data provide profiles of proteins and pathways needed for recovery after both genotoxic and non-genotoxic compounds. In addition, a novel role for aromatic amino acids in the recovery after treatment with oxidizing agents was suggested. The role of aromatic acids was further validated; the quinone subgroup of oxidizing agents were extremely toxic in cells where tryptophan biosynthesis was compromised.

DNA Barcoding of Zooplankton in the Hampton Roads Area: A Biodiversity Assessment

NASA Astrophysics Data System (ADS)

Salcedo, A.; Rodríguez, Á. E.; Gibson, D. M.

2016-02-01

The study of zooplankton biodiversity and distribution is crucial to understand oceanic ecosystems and anticipate the effects of climate change. Previously, identification of zooplankton relied in morphological identification employed by expert taxonomists. DNA barcoding, a technique that uses the mitochondrial DNA (mtDNA) Cytochrome Oxidase 1 (CO1) gene is widely used for taxonomic identification. Thus, this molecular technique will be used to begin a detailed characterization of zooplankton diversity, abundance and community structure in the Hampton Roads Area (HRA). Stations 1 (Jones Creek) and 3 (lower Chesapeake Bay) were sampled in June 19, 2015. Stations 1, 2 (James River), and 3 were sampled in September 2015. Zooplankton samples were collected in triplicates with a 0.5m, 200 µm mesh net. Physical parameters (dissolved oxygen, salinity, temperature and, water transparency) were measured. Species identified as Opistonema oglinum (Atlantic Thread Herring) and Paracalanus parvus copepods were found at station 3; Anchoa mitchilli and Acartia tonsa copepods were found at stations 1 and 3. This study indicates that mtDNA-CO1 barcoding is suitable to identify zooplankton to the species level and helps validate DNA barcoding as a faster, more accurate taxonomic approach. The long term objective of this project is to provide a comprehensive assessment of zooplankton in the HRA and to generate a reference record for broad monitoring programs; vital for a better understanding and management of ecologically and commercially important species.

A DNA Barcoding Approach to Characterize Pollen Collected by Honeybees

PubMed Central

Bruni, Ilaria; Scaccabarozzi, Daniela; Sandionigi, Anna; Barbuto, Michela; Casiraghi, Maurizio; Labra, Massimo

2014-01-01

In the present study, we investigated DNA barcoding effectiveness to characterize honeybee pollen pellets, a food supplement largely used for human nutrition due to its therapeutic properties. We collected pollen pellets using modified beehives placed in three zones within an alpine protected area (Grigna Settentrionale Regional Park, Italy). A DNA barcoding reference database, including rbcL and trnH-psbA sequences from 693 plant species (104 sequenced in this study) was assembled. The database was used to identify pollen collected from the hives. Fifty-two plant species were identified at the molecular level. Results suggested rbcL alone could not distinguish among congeneric plants; however, psbA-trnH identified most of the pollen samples at the species level. Substantial variability in pollen composition was observed between the highest elevation locality (Alpe Moconodeno), characterized by arid grasslands and a rocky substrate, and the other two sites (Cornisella and Ortanella) at lower altitudes. Pollen from Ortanella and Cornisella showed the presence of typical deciduous forest species; however in samples collected at Ortanella, pollen of the invasive Lonicera japonica, and the ornamental Pelargonium x hortorum were observed. Our results indicated pollen composition was largely influenced by floristic local biodiversity, plant phenology, and the presence of alien flowering species. Therefore, pollen molecular characterization based on DNA barcoding might serve useful to beekeepers in obtaining honeybee products with specific nutritional or therapeutic characteristics desired by food market demands. PMID:25296114

mPUMA: a computational approach to microbiota analysis by de novo assembly of operational taxonomic units based on protein-coding barcode sequences.

PubMed

Links, Matthew G; Chaban, Bonnie; Hemmingsen, Sean M; Muirhead, Kevin; Hill, Janet E

2013-08-15

Formation of operational taxonomic units (OTU) is a common approach to data aggregation in microbial ecology studies based on amplification and sequencing of individual gene targets. The de novo assembly of OTU sequences has been recently demonstrated as an alternative to widely used clustering methods, providing robust information from experimental data alone, without any reliance on an external reference database. Here we introduce mPUMA (microbial Profiling Using Metagenomic Assembly, http://mpuma.sourceforge.net), a software package for identification and analysis of protein-coding barcode sequence data. It was developed originally for Cpn60 universal target sequences (also known as GroEL or Hsp60). Using an unattended process that is independent of external reference sequences, mPUMA forms OTUs by DNA sequence assembly and is capable of tracking OTU abundance. mPUMA processes microbial profiles both in terms of the direct DNA sequence as well as in the translated amino acid sequence for protein coding barcodes. By forming OTUs and calculating abundance through an assembly approach, mPUMA is capable of generating inputs for several popular microbiota analysis tools. Using SFF data from sequencing of a synthetic community of Cpn60 sequences derived from the human vaginal microbiome, we demonstrate that mPUMA can faithfully reconstruct all expected OTU sequences and produce compositional profiles consistent with actual community structure. mPUMA enables analysis of microbial communities while empowering the discovery of novel organisms through OTU assembly.

Assessment of the Authenticity of Herbal Dietary Supplements: Comparison of Chemical and DNA Barcoding Methods.

PubMed

Pawar, Rahul S; Handy, Sara M; Cheng, Raymond; Shyong, Nicole; Grundel, Erich

2017-07-01

About 7 % of the U. S. population reports using botanical dietary supplements. Increased use of such supplements has led to discussions related to their authenticity and quality. Reports of adulteration with substandard materials or pharmaceuticals are of concern because such substitutions, whether inadvertent or deliberate, may reduce the efficacy of specific botanicals or lead to adverse events. Methods for verifying the identity of botanicals include macroscopic and microscopic examinations, chemical analysis, and DNA-based methods including DNA barcoding. Macroscopic and microscopic examinations may fail when a supplement consists of botanicals that have been processed beyond the ability to provide morphological characterizations. Chemical analysis of specific marker compounds encounters problems when these compounds are not distinct to a given species or when purified reference standards are not available. Recent investigations describing DNA barcoding analysis of botanical dietary supplements have raised concerns about the authenticity of the supplements themselves as well as the appropriateness of using DNA barcoding techniques with finished botanical products. We collected 112 market samples of frequently consumed botanical dietary supplements of ginkgo, soy, valerian, yohimbe, and St. John's wort and analyzed each for specific chemical markers (i.e., flavonol glycosides, total isoflavones, total valerenic acids, yohimbine, and hypericins, respectively). We used traditional DNA barcoding techniques targeting the nuclear ITS2 gene and the chloroplast gene psb A- trn H on the same samples to determine the presence of DNA of the labelled ingredient. We compared the results obtained by both methods to assess the contribution of each in determining the identity of the samples. Georg Thieme Verlag KG Stuttgart · New York.

Redefining reference in an academic health sciences library: planning for change.

PubMed

Gray, S A; Brower, S; Munger, H; Start, A; White, P

2001-01-01

Deciding that changes in the pattern of questions at the reference desk required focused consideration, the reference librarians at the Health Sciences Library of the University at Buffalo held a planning retreat. Technology-induced changes in the information-seeking behavior and reference needs of the library's clientele caused a reassessment of how these needs could best be met and what is the best use of librarians' time. The librarians considered current trends in reference in other academic libraries, the specific needs of the clientele of the Health Sciences Library, and the strengths and expertise of the library staff. The results of this structured discussion produced ideas for redefining reference to provide customized services for the clients and environment.

Tropical Plant–Herbivore Networks: Reconstructing Species Interactions Using DNA Barcodes

PubMed Central

García-Robledo, Carlos; Erickson, David L.; Staines, Charles L.; Erwin, Terry L.; Kress, W. John

2013-01-01

Plants and their associated insect herbivores, represent more than 50% of all known species on earth. The first step in understanding the mechanisms generating and maintaining this important component of biodiversity is to identify plant-herbivore associations. In this study we determined insect-host plant associations for an entire guild of insect herbivores using plant DNA extracted from insect gut contents. Over two years, in a tropical rain forest in Costa Rica (La Selva Biological Station), we recorded the full diet breadth of rolled-leaf beetles, a group of herbivores that feed on plants in the order Zingiberales. Field observations were used to determine the accuracy of diet identifications using a three-locus DNA barcode (rbcL, trnH-psbA and ITS2). Using extraction techniques for ancient DNA, we obtained high-quality sequences for two of these loci from gut contents (rbcL and ITS2). Sequences were then compared to a comprehensive DNA barcode library of the Zingiberales. The rbcL locus identified host plants to family (success/sequence = 58.8%) and genus (success/sequence = 47%). For all Zingiberales except Heliconiaceae, ITS2 successfully identified host plants to genus (success/sequence = 67.1%) and species (success/sequence = 61.6%). Kindt’s sampling estimates suggest that by collecting ca. four individuals representing each plant-herbivore interaction, 99% of all host associations included in this study can be identified to genus. For plants that amplified ITS2, 99% of the hosts can be identified to species after collecting at least four individuals representing each interaction. Our study demonstrates that host plant identifications at the species-level using DNA barcodes are feasible, cost-effective, and reliable, and that reconstructing plant-herbivore networks with these methods will become the standard for a detailed understanding of these interactions. PMID:23308128

The whole genome sequences and experimentally phased haplotypes of over 100 personal genomes.

PubMed

Mao, Qing; Ciotlos, Serban; Zhang, Rebecca Yu; Ball, Madeleine P; Chin, Robert; Carnevali, Paolo; Barua, Nina; Nguyen, Staci; Agarwal, Misha R; Clegg, Tom; Connelly, Abram; Vandewege, Ward; Zaranek, Alexander Wait; Estep, Preston W; Church, George M; Drmanac, Radoje; Peters, Brock A

2016-10-11

Since the completion of the Human Genome Project in 2003, it is estimated that more than 200,000 individual whole human genomes have been sequenced. A stunning accomplishment in such a short period of time. However, most of these were sequenced without experimental haplotype data and are therefore missing an important aspect of genome biology. In addition, much of the genomic data is not available to the public and lacks phenotypic information. As part of the Personal Genome Project, blood samples from 184 participants were collected and processed using Complete Genomics' Long Fragment Read technology. Here, we present the experimental whole genome haplotyping and sequencing of these samples to an average read coverage depth of 100X. This is approximately three-fold higher than the read coverage applied to most whole human genome assemblies and ensures the highest quality results. Currently, 114 genomes from this dataset are freely available in the GigaDB repository and are associated with rich phenotypic data; the remaining 70 should be added in the near future as they are approved through the PGP data release process. For reproducibility analyses, 20 genomes were sequenced at least twice using independent LFR barcoded libraries. Seven genomes were also sequenced using Complete Genomics' standard non-barcoded library process. In addition, we report 2.6 million high-quality, rare variants not previously identified in the Single Nucleotide Polymorphisms database or the 1000 Genomes Project Phase 3 data. These genomes represent a unique source of haplotype and phenotype data for the scientific community and should help to expand our understanding of human genome evolution and function.

Forensic botany II, DNA barcode for land plants: Which markers after the international agreement?

PubMed

Ferri, G; Corradini, B; Ferrari, F; Santunione, A L; Palazzoli, F; Alu', M

2015-03-01

The ambitious idea of using a short piece of DNA for large-scale species identification (DNA barcoding) is already a powerful tool for scientists and the application of this standard technique seems promising in a range of fields including forensic genetics. While DNA barcoding enjoyed a remarkable success for animal identification through cytochrome c oxidase I (COI) analysis, the attempts to identify a single barcode for plants remained a vain hope for a longtime. From the beginning, the Consortium for the Barcode of Life (CBOL) showed a lack of agreement on a core plant barcode, reflecting the diversity of viewpoints. Different research groups advocated various markers with divergent set of criteria until the recent publication by the CBOL-Plant Working Group. After a four-year effort, in 2009 the International Team concluded to agree on standard markers promoting a multilocus solution (rbcL and matK), with 70-75% of discrimination to the species level. In 2009 our group firstly proposed the broad application of DNA barcoding principles as a tool for identification of trace botanical evidence through the analysis of two chloroplast loci (trnH-psbA and trnL-trnF) in plant species belonging to local flora. Difficulties and drawbacks that were encountered included a poor coverage of species in specific databases and the lack of authenticated reference sequences for the selected markers. Successful preliminary results were obtained providing an approach to progressively identify unknown plant specimens to a given taxonomic rank, usable by any non-specialist botanist or in case of a shortage of taxonomic expertise. Now we considered mandatory to update and to compare our previous findings with the new selected plastid markers (matK+rbcL), taking into account forensic requirements. Features of all the four loci (the two previously analyzed trnH-psbA+trnL-trnF and matK+rbcL) were compared singly and in multilocus solutions to assess the most suitable combination for forensic botany. Based on obtained results, we recommend the adoption of a two-locus combination with rbcL+trnH-psbA plastid markers, which currently best satisfies forensic needs for botanical species identification. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Self-registering spread-spectrum barcode method

DOEpatents

Cummings, Eric B.; Even Jr., William R.

2004-11-09

A novel spread spectrum barcode methodology is disclosed that allows a barcode to be read in its entirety even when a significant fraction or majority of the barcode is obscured. The barcode methodology makes use of registration or clocking information that is distributed along with the encoded user data across the barcode image. This registration information allows for the barcode image to be corrected for imaging distortion such as zoom, rotation, tilt, curvature, and perspective.

Re-cataloging Joint Astronomy Centre (JAC) Library Book Collection

NASA Astrophysics Data System (ADS)

Lucas, A.; Zhang, X.

2007-10-01

The Joint Astronomy Centre operates two telescopes: the James Clerk Maxwell Telescope and the United Kingdom Infrared Telescope. In the JAC's 25-year history, their library was maintained by a number of staff ranging from scientists to student assistants. This resulted in an inconsistent and incomplete catalog as well as a mixture of typed, hand written, and inaccurate call number labels. Further complicating the situation was a backlog of un-cataloged books. In the process of improving the library system, it became obvious that the entire book collection needed to be re-cataloged and re-labeled. Readerware proved to be an inexpensive and efficient tool for this project. The software allows for the scanning of barcodes or the manual input of ISBNs, LCCNs and UPCs. It then retrieves the cataloging records from a number of pre-selected websites. The merged information is then stored in a database that can be manipulated to perform tasks such as printing call number labels. Readerware is also ideal for copy cataloging and has become an indispensable tool in maintaining the JAC's collection of books.

Hit-Validation Methodologies for Ligands Isolated from DNA-Encoded Chemical Libraries.

PubMed

Zimmermann, Gunther; Li, Yizhou; Rieder, Ulrike; Mattarella, Martin; Neri, Dario; Scheuermann, Jörg

2017-05-04

DNA-encoded chemical libraries (DECLs) are large collections of compounds linked to DNA fragments, serving as amplifiable barcodes, which can be screened on target proteins of interest. In typical DECL selections, preferential binders are identified by high-throughput DNA sequencing, by comparing their frequency before and after the affinity capture step. Hits identified in this procedure need to be confirmed, by resynthesis and by performing affinity measurements. In this article we present new methods based on hybridization of oligonucleotide conjugates with fluorescently labeled complementary oligonucleotides; these facilitate the determination of affinity constants and kinetic dissociation constants. The experimental procedures were demonstrated with acetazolamide, a binder to carbonic anhydrase IX with a dissociation constant in the nanomolar range. The detection of binding events was compatible not only with fluorescence polarization methodologies, but also with Alphascreen technology and with microscale thermophoresis. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Reconstructing a herbivore’s diet using a novel rbcL DNA mini-barcode for plants

USGS Publications Warehouse

Erickson, David L.; Reed, Elizabeth; Ramachandran, Padmini; Bourg, Norman; McShea, William J.; Ottesen, Andrea

2017-01-01

Next Generation Sequencing and the application of metagenomic analyses can be used to answer questions about animal diet choice and study the consequences of selective foraging by herbivores. The quantification of herbivore diet choice with respect to native versus exotic plant species is particularly relevant given concerns of invasive species establishment and their effects on ecosystems. While increased abundance of white-tailed deer (Odocoileus virginianus) appears to correlate with increased incidence of invasive plant species, data supporting a causal link is scarce. We used a metabarcoding approach (PCR amplicons of the plant rbcL gene) to survey the diet of white-tailed deer (fecal samples), from a forested site in Warren County, Virginia with a comprehensive plant species inventory and corresponding reference collection of plant barcode and chloroplast sequences. We sampled fecal pellet piles and extracted DNA from 12 individual deer in October 2014. These samples were compared to a reference DNA library of plant species collected within the study area. For 72 % of the amplicons, we were able to assign taxonomy at the species level, which provides for the first time—sufficient taxonomic resolution to quantify the relative frequency at which native and exotic plant species are being consumed by white-tailed deer. For each of the 12 individual deer we collected three subsamples from the same fecal sample, resulting in sequencing 36 total samples. Using Qiime, we quantified the plant DNA found in all 36 samples, and found that variance within samples was less than variance between samples (F = 1.73, P = 0.004), indicating additional subsamples may not be necessary. Species level diversity ranged from 60 to 93 OTUs per individual and nearly 70 % of all plant sequences recovered were from native plant species. The number of species detected did reduce significantly (range 4–12) when we excluded species whose OTU composed <1 % of each sample’s total. When compared to the abundance of native and non-natives plants inventoried in the local community, our results support the observation that white-tailed deer have strong foraging preferences, but these preferences were not consistent for species in either class. Deer forage behaviour may favour some exotic species, but not all.

Reconstructing a herbivore's diet using a novel rbcL DNA mini-barcode for plants.

PubMed

Erickson, David L; Reed, Elizabeth; Ramachandran, Padmini; Bourg, Norman A; McShea, William J; Ottesen, Andrea

2017-05-01

Next Generation Sequencing and the application of metagenomic analyses can be used to answer questions about animal diet choice and study the consequences of selective foraging by herbivores. The quantification of herbivore diet choice with respect to native versus exotic plant species is particularly relevant given concerns of invasive species establishment and their effects on ecosystems. While increased abundance of white-tailed deer ( Odocoileus virginianus ) appears to correlate with increased incidence of invasive plant species, data supporting a causal link is scarce. We used a metabarcoding approach (PCR amplicons of the plant rbc L gene) to survey the diet of white-tailed deer (fecal samples), from a forested site in Warren County, Virginia with a comprehensive plant species inventory and corresponding reference collection of plant barcode and chloroplast sequences. We sampled fecal pellet piles and extracted DNA from 12 individual deer in October 2014. These samples were compared to a reference DNA library of plant species collected within the study area. For 72 % of the amplicons, we were able to assign taxonomy at the species level, which provides for the first time-sufficient taxonomic resolution to quantify the relative frequency at which native and exotic plant species are being consumed by white-tailed deer. For each of the 12 individual deer we collected three subsamples from the same fecal sample, resulting in sequencing 36 total samples. Using Qiime, we quantified the plant DNA found in all 36 samples, and found that variance within samples was less than variance between samples ( F  = 1.73, P  = 0.004), indicating additional subsamples may not be necessary. Species level diversity ranged from 60 to 93 OTUs per individual and nearly 70 % of all plant sequences recovered were from native plant species. The number of species detected did reduce significantly (range 4-12) when we excluded species whose OTU composed <1 % of each sample's total. When compared to the abundance of native and non-natives plants inventoried in the local community, our results support the observation that white-tailed deer have strong foraging preferences, but these preferences were not consistent for species in either class. Deer forage behaviour may favour some exotic species, but not all.

Reconstructing a herbivore’s diet using a novel rbcL DNA mini-barcode for plants

PubMed Central

Erickson, David L.; Reed, Elizabeth; Ramachandran, Padmini; Bourg, Norman A.; Ottesen, Andrea

2017-01-01

Abstract Next Generation Sequencing and the application of metagenomic analyses can be used to answer questions about animal diet choice and study the consequences of selective foraging by herbivores. The quantification of herbivore diet choice with respect to native versus exotic plant species is particularly relevant given concerns of invasive species establishment and their effects on ecosystems. While increased abundance of white-tailed deer (Odocoileus virginianus) appears to correlate with increased incidence of invasive plant species, data supporting a causal link is scarce. We used a metabarcoding approach (PCR amplicons of the plant rbcL gene) to survey the diet of white-tailed deer (fecal samples), from a forested site in Warren County, Virginia with a comprehensive plant species inventory and corresponding reference collection of plant barcode and chloroplast sequences. We sampled fecal pellet piles and extracted DNA from 12 individual deer in October 2014. These samples were compared to a reference DNA library of plant species collected within the study area. For 72 % of the amplicons, we were able to assign taxonomy at the species level, which provides for the first time—sufficient taxonomic resolution to quantify the relative frequency at which native and exotic plant species are being consumed by white-tailed deer. For each of the 12 individual deer we collected three subsamples from the same fecal sample, resulting in sequencing 36 total samples. Using Qiime, we quantified the plant DNA found in all 36 samples, and found that variance within samples was less than variance between samples (F = 1.73, P = 0.004), indicating additional subsamples may not be necessary. Species level diversity ranged from 60 to 93 OTUs per individual and nearly 70 % of all plant sequences recovered were from native plant species. The number of species detected did reduce significantly (range 4–12) when we excluded species whose OTU composed <1 % of each sample’s total. When compared to the abundance of native and non-natives plants inventoried in the local community, our results support the observation that white-tailed deer have strong foraging preferences, but these preferences were not consistent for species in either class. Deer forage behaviour may favour some exotic species, but not all. PMID:28533898

Locating and decoding barcodes in fuzzy images captured by smart phones

NASA Astrophysics Data System (ADS)

Deng, Wupeng; Hu, Jiwei; Liu, Quan; Lou, Ping

2017-07-01

With the development of barcodes for commercial use, people's requirements for detecting barcodes by smart phone become increasingly pressing. The low quality of barcode image captured by mobile phone always affects the decoding and recognition rates. This paper focuses on locating and decoding EAN-13 barcodes in fuzzy images. We present a more accurate locating algorithm based on segment length and high fault-tolerant rate algorithm for decoding barcodes. Unlike existing approaches, location algorithm is based on the edge segment length of EAN -13 barcodes, while our decoding algorithm allows the appearance of fuzzy region in barcode image. Experimental results are performed on damaged, contaminated and scratched digital images, and provide a quite promising result for EAN -13 barcode location and decoding.

A single mini-barcode test to screen for Australian mammalian predators from environmental samples

PubMed Central

MacDonald, Anna J; Sarre, Stephen D

2017-01-01

Abstract Identification of species from trace samples is now possible through the comparison of diagnostic DNA fragments against reference DNA sequence databases. DNA detection of animals from non-invasive samples, such as predator faeces (scats) that contain traces of DNA from their species of origin, has proved to be a valuable tool for the management of elusive wildlife. However, application of this approach can be limited by the availability of appropriate genetic markers. Scat DNA is often degraded, meaning that longer DNA sequences, including standard DNA barcoding markers, are difficult to recover. Instead, targeted short diagnostic markers are required to serve as diagnostic mini-barcodes. The mitochondrial genome is a useful source of such trace DNA markers because it provides good resolution at the species level and occurs in high copy numbers per cell. We developed a mini-barcode based on a short (178 bp) fragment of the conserved 12S ribosomal ribonucleic acid mitochondrial gene sequence, with the goal of discriminating amongst the scats of large mammalian predators of Australia. We tested the sensitivity and specificity of our primers and can accurately detect and discriminate amongst quolls, cats, dogs, foxes, and devils from trace DNA samples. Our approach provides a cost-effective, time-efficient, and non-invasive tool that enables identification of all 8 medium-large mammal predators in Australia, including native and introduced species, using a single test. With modification, this approach is likely to be of broad applicability elsewhere. PMID:28810700

Evaluating a Chat Reference Service at the University of South Alabama's Baugh Biomedical Library

ERIC Educational Resources Information Center

Clanton, Clista C.; Staggs, Geneva B.; Williams, Thomas L.

2006-01-01

The University of South Alabama's Baugh Biomedical Library recently initiated a chat reference service targeted at distance education students in the biomedical sciences. After one year of service, the library conducted an evaluation of the chat reference to assess the success of this mode of reference service. Both traditional reference and…

Use of rbcL and trnL-F as a Two-Locus DNA Barcode for Identification of NW-European Ferns: An Ecological Perspective

PubMed Central

de Groot, G. Arjen; During, Heinjo J.; Maas, Jan W.; Schneider, Harald; Vogel, Johannes C.; Erkens, Roy H. J.

2011-01-01

Although consensus has now been reached on a general two-locus DNA barcode for land plants, the selected combination of markers (rbcL + matK) is not applicable for ferns at the moment. Yet especially for ferns, DNA barcoding is potentially of great value since fern gametophytes—while playing an essential role in fern colonization and reproduction—generally lack the morphological complexity for morphology-based identification and have therefore been underappreciated in ecological studies. We evaluated the potential of a combination of rbcL with a noncoding plastid marker, trnL-F, to obtain DNA-identifications for fern species. A regional approach was adopted, by creating a reference database of trusted rbcL and trnL-F sequences for the wild-occurring homosporous ferns of NW-Europe. A combination of parsimony analyses and distance-based analyses was performed to evaluate the discriminatory power of the two-region barcode. DNA was successfully extracted from 86 tiny fern gametophytes and was used as a test case for the performance of DNA-based identification. Primer universality proved high for both markers. Based on the combined rbcL + trnL-F dataset, all genera as well as all species with non-equal chloroplast genomes formed their own well supported monophyletic clade, indicating a high discriminatory power. Interspecific distances were larger than intraspecific distances for all tested taxa. Identification tests on gametophytes showed a comparable result. All test samples could be identified to genus level, species identification was well possible unless they belonged to a pair of Dryopteris species with completely identical chloroplast genomes. Our results suggest a high potential of the combined use of rbcL and trnL-F as a two-locus cpDNA barcode for identification of fern species. A regional approach may be preferred for ecological tests. We here offer such a ready-to-use barcoding approach for ferns, which opens the way for answering a whole range of questions previously unaddressed in fern gametophyte ecology. PMID:21298108

Fast lossless compression via cascading Bloom filters

PubMed Central

2014-01-01

Background Data from large Next Generation Sequencing (NGS) experiments present challenges both in terms of costs associated with storage and in time required for file transfer. It is sometimes possible to store only a summary relevant to particular applications, but generally it is desirable to keep all information needed to revisit experimental results in the future. Thus, the need for efficient lossless compression methods for NGS reads arises. It has been shown that NGS-specific compression schemes can improve results over generic compression methods, such as the Lempel-Ziv algorithm, Burrows-Wheeler transform, or Arithmetic Coding. When a reference genome is available, effective compression can be achieved by first aligning the reads to the reference genome, and then encoding each read using the alignment position combined with the differences in the read relative to the reference. These reference-based methods have been shown to compress better than reference-free schemes, but the alignment step they require demands several hours of CPU time on a typical dataset, whereas reference-free methods can usually compress in minutes. Results We present a new approach that achieves highly efficient compression by using a reference genome, but completely circumvents the need for alignment, affording a great reduction in the time needed to compress. In contrast to reference-based methods that first align reads to the genome, we hash all reads into Bloom filters to encode, and decode by querying the same Bloom filters using read-length subsequences of the reference genome. Further compression is achieved by using a cascade of such filters. Conclusions Our method, called BARCODE, runs an order of magnitude faster than reference-based methods, while compressing an order of magnitude better than reference-free methods, over a broad range of sequencing coverage. In high coverage (50-100 fold), compared to the best tested compressors, BARCODE saves 80-90% of the running time while only increasing space slightly. PMID:25252952

Fast lossless compression via cascading Bloom filters.

PubMed

Rozov, Roye; Shamir, Ron; Halperin, Eran

2014-01-01

Data from large Next Generation Sequencing (NGS) experiments present challenges both in terms of costs associated with storage and in time required for file transfer. It is sometimes possible to store only a summary relevant to particular applications, but generally it is desirable to keep all information needed to revisit experimental results in the future. Thus, the need for efficient lossless compression methods for NGS reads arises. It has been shown that NGS-specific compression schemes can improve results over generic compression methods, such as the Lempel-Ziv algorithm, Burrows-Wheeler transform, or Arithmetic Coding. When a reference genome is available, effective compression can be achieved by first aligning the reads to the reference genome, and then encoding each read using the alignment position combined with the differences in the read relative to the reference. These reference-based methods have been shown to compress better than reference-free schemes, but the alignment step they require demands several hours of CPU time on a typical dataset, whereas reference-free methods can usually compress in minutes. We present a new approach that achieves highly efficient compression by using a reference genome, but completely circumvents the need for alignment, affording a great reduction in the time needed to compress. In contrast to reference-based methods that first align reads to the genome, we hash all reads into Bloom filters to encode, and decode by querying the same Bloom filters using read-length subsequences of the reference genome. Further compression is achieved by using a cascade of such filters. Our method, called BARCODE, runs an order of magnitude faster than reference-based methods, while compressing an order of magnitude better than reference-free methods, over a broad range of sequencing coverage. In high coverage (50-100 fold), compared to the best tested compressors, BARCODE saves 80-90% of the running time while only increasing space slightly.

Reference Materials and Services for a Small Hospital Library. 5th Revised Edition.

ERIC Educational Resources Information Center

Kesti, Julie, Comp.; Graham, Elaine, Comp.

This manual suggests and describes recommended reference services and sources for a small hospital library. Focusing on reference services, the first section includes information on ready-reference services; bibliographic search services, including taking and processing a request for a bibliography, National Library of Medicine literature…

Factors Influencing Competence and Performance of Reference Librarians in Academic Libraries in Nigeria.

ERIC Educational Resources Information Center

Omoniyi, Joseph O.

2002-01-01

Reports on research that focused on competence and performance of reference libraries in academic libraries in Nigeria. Includes recommendations for improving competence and performance, including the reference process; staffing and other resources; education for reference librarians; knowledge of modern technology; and emotional stability and…

Raising the Bar or Training Library Technicians To Assume Reference Responsibilities.

ERIC Educational Resources Information Center

Brandys, Barbara; Daghita, Joan; Whitmore, Susan

This paper reports on a program at the National Institutes of Health (NIH) Library that was instituted to train library technicians to work at the Information Desk as Reference Assistants; the objectives of the program were to train library technicians to become reference assistants, to free up librarians' time for new work assignments, and to…

The Implications of Library Anxiety for Academic Reference Services: A Review of the Literature

ERIC Educational Resources Information Center

Carlile, Heather

2007-01-01

Academic reference librarians continually observe that many students are embarrassed about not knowing how to use the library and are reluctant to approach the reference desk. The theory of library anxiety offers an explanation, proposing that a fear of being in and using libraries serves as a psychological barrier, hindering many university…

A Survey of the Usability of Digital Reference Services on Academic Health Science Library Web Sites

ERIC Educational Resources Information Center

Dee, Cheryl; Allen, Maryellen

2006-01-01

Reference interactions with patrons in a digital library environment using digital reference services (DRS) has become widespread. However, such services in many libraries appear to be underutilized. A study surveying the ease and convenience of such services for patrons in over 100 academic health science library Web sites suggests that…

Horse Racing at the Library: How One Library System Increased the Usage of Some of Its Online Databases

ERIC Educational Resources Information Center

Kurhan, Scott H.; Griffing, Elizabeth A.

2011-01-01

Reference services in public libraries are changing dramatically. The Internet, online databases, and shrinking budgets are all making it necessary for non-traditional reference staff to become familiar with online reference tools. Recognizing the need for cross-training, Chesapeake Public Library (CPL) developed a program called the Database…

Eliminating traditional reference services in an academic health sciences library: a case study

PubMed Central

Schulte, Stephanie J

2011-01-01

Question: How were traditional librarian reference desk services successfully eliminated at one health sciences library? Setting: The analysis was done at an academic health sciences library at a major research university. Method: A gap analysis was performed, evaluating changes in the first eleven months through analysis of reference transaction and instructional session data. Main Results: Substantial increases were seen in the overall number of specialized reference transactions and those conducted by librarians lasting more than thirty minutes. The number of reference transactions overall increased after implementing the new model. Several new small-scale instructional initiatives began, though perhaps not directly related to the new model. Conclusion: Traditional reference desk services were eliminated at one academic health sciences library without negative impact on reference and instructional statistics. Eliminating ties to the confines of the physical library due to staffing reference desk hours removed one significant barrier to a more proactive liaison program. PMID:22022221

Efficient alignment-free DNA barcode analytics.

PubMed

Kuksa, Pavel; Pavlovic, Vladimir

2009-11-10

In this work we consider barcode DNA analysis problems and address them using alternative, alignment-free methods and representations which model sequences as collections of short sequence fragments (features). The methods use fixed-length representations (spectrum) for barcode sequences to measure similarities or dissimilarities between sequences coming from the same or different species. The spectrum-based representation not only allows for accurate and computationally efficient species classification, but also opens possibility for accurate clustering analysis of putative species barcodes and identification of critical within-barcode loci distinguishing barcodes of different sample groups. New alignment-free methods provide highly accurate and fast DNA barcode-based identification and classification of species with substantial improvements in accuracy and speed over state-of-the-art barcode analysis methods. We evaluate our methods on problems of species classification and identification using barcodes, important and relevant analytical tasks in many practical applications (adverse species movement monitoring, sampling surveys for unknown or pathogenic species identification, biodiversity assessment, etc.) On several benchmark barcode datasets, including ACG, Astraptes, Hesperiidae, Fish larvae, and Birds of North America, proposed alignment-free methods considerably improve prediction accuracy compared to prior results. We also observe significant running time improvements over the state-of-the-art methods. Our results show that newly developed alignment-free methods for DNA barcoding can efficiently and with high accuracy identify specimens by examining only few barcode features, resulting in increased scalability and interpretability of current computational approaches to barcoding.

Transient partial permeabilization with saponin enables cellular barcoding prior to surface marker staining1

PubMed Central

Behbehani, Gregory K.; Thom, Colin; Zunder, Eli R.; Finck, Rachel; Gaudilliere, Brice; Fragiadakis, Gabriela K.; Fantl, Wendy J.; Nolan, Garry P.

2015-01-01

Fluorescent cellular barcoding and mass-tag cellular barcoding are cytometric methods that enable high sample throughput, minimize inter-sample variation, and reduce reagent consumption. Previously employed barcoding protocols require that barcoding be performed after surface marker staining, complicating combining the technique with measurement of alcohol-sensitive surface epitopes. This report describes a method of barcoding fixed cells after a transient partial permeabilization with 0.02% saponin that results in efficient and consistent barcode staining with fluorescent or mass-tagged reagents while preserving surface marker staining. This approach simplifies barcoding protocols and allows direct comparison of surface marker staining of multiple samples without concern for variations in the antibody cocktail volume, antigen-antibody ratio, or machine sensitivity. Using this protocol, cellular barcoding can be used to reliably detect subtle differences in surface marker expression. PMID:25274027

DNA barcoding a nightmare taxon: assessing barcode index numbers and barcode gaps for sweat bees.

PubMed

Gibbs, Jason

2018-01-01

There is an ongoing campaign to DNA barcode the world's >20 000 bee species. Recent revisions of Lasioglossum (Dialictus) (Hymenoptera: Halictidae) for Canada and the eastern United States were completed using integrative taxonomy. DNA barcode data from 110 species of L. (Dialictus) are examined for their value in identification and discovering additional taxonomic diversity. Specimen identification success was estimated using the best close match method. Error rates were 20% relative to current taxonomic understanding. Barcode Index Numbers (BINs) assigned using Refined Single Linkage Analysis (RESL) and barcode gaps using the Automatic Barcode Gap Discovery (ABGD) method were also assessed. RESL was incongruent for 44.5% of species, although some cryptic diversity may exist. Forty-three of 110 species were part of merged BINs with multiple species. The barcode gap is non-existent for the data set as a whole and ABGD showed levels of discordance similar to the RESL. The viridatum species-group is particularly problematic, so that DNA barcodes alone would be misleading for species delimitation and specimen identification. Character-based methods using fixed nucleotide substitutions could improve specimen identification success in some cases. The use of DNA barcoding for species discovery for standard taxonomic practice in the absence of a well-defined barcode gap is discussed.

Multiplexed microsatellite recovery using massively parallel sequencing

USGS Publications Warehouse

Jennings, T.N.; Knaus, B.J.; Mullins, T.D.; Haig, S.M.; Cronn, R.C.

2011-01-01

Conservation and management of natural populations requires accurate and inexpensive genotyping methods. Traditional microsatellite, or simple sequence repeat (SSR), marker analysis remains a popular genotyping method because of the comparatively low cost of marker development, ease of analysis and high power of genotype discrimination. With the availability of massively parallel sequencing (MPS), it is now possible to sequence microsatellite-enriched genomic libraries in multiplex pools. To test this approach, we prepared seven microsatellite-enriched, barcoded genomic libraries from diverse taxa (two conifer trees, five birds) and sequenced these on one lane of the Illumina Genome Analyzer using paired-end 80-bp reads. In this experiment, we screened 6.1 million sequences and identified 356958 unique microreads that contained di- or trinucleotide microsatellites. Examination of four species shows that our conversion rate from raw sequences to polymorphic markers compares favourably to Sanger- and 454-based methods. The advantage of multiplexed MPS is that the staggering capacity of modern microread sequencing is spread across many libraries; this reduces sample preparation and sequencing costs to less than $400 (USD) per species. This price is sufficiently low that microsatellite libraries could be prepared and sequenced for all 1373 organisms listed as 'threatened' and 'endangered' in the United States for under $0.5M (USD).

Enterprise Reference Library

NASA Technical Reports Server (NTRS)

Bickham, Grandin; Saile, Lynn; Havelka, Jacque; Fitts, Mary

2011-01-01

Introduction: Johnson Space Center (JSC) offers two extensive libraries that contain journals, research literature and electronic resources. Searching capabilities are available to those individuals residing onsite or through a librarian s search. Many individuals have rich collections of references, but no mechanisms to share reference libraries across researchers, projects, or directorates exist. Likewise, information regarding which references are provided to which individuals is not available, resulting in duplicate requests, redundant labor costs and associated copying fees. In addition, this tends to limit collaboration between colleagues and promotes the establishment of individual, unshared silos of information The Integrated Medical Model (IMM) team has utilized a centralized reference management tool during the development, test, and operational phases of this project. The Enterprise Reference Library project expands the capabilities developed for IMM to address the above issues and enhance collaboration across JSC. Method: After significant market analysis for a multi-user reference management tool, no available commercial tool was found to meet this need, so a software program was built around a commercial tool, Reference Manager 12 by The Thomson Corporation. A use case approach guided the requirements development phase. The premise of the design is that individuals use their own reference management software and export to SharePoint when their library is incorporated into the Enterprise Reference Library. This results in a searchable user-specific library application. An accompanying share folder will warehouse the electronic full-text articles, which allows the global user community to access full -text articles. Discussion: An enterprise reference library solution can provide a multidisciplinary collection of full text articles. This approach improves efficiency in obtaining and storing reference material while greatly reducing labor, purchasing and duplication costs. Most importantly, increasing collaboration across research groups provides unprecedented access to information relevant to NASA s mission. Conclusion: This project is an expansion and cost-effective leveraging of the existing JSC centralized library. Adding key word and author search capabilities and an alert function for notifications about new articles, based on users profiles, represent examples of future enhancements.

Constructing DNA Barcode Sets Based on Particle Swarm Optimization.

PubMed

Wang, Bin; Zheng, Xuedong; Zhou, Shihua; Zhou, Changjun; Wei, Xiaopeng; Zhang, Qiang; Wei, Ziqi

2018-01-01

Following the completion of the human genome project, a large amount of high-throughput bio-data was generated. To analyze these data, massively parallel sequencing, namely next-generation sequencing, was rapidly developed. DNA barcodes are used to identify the ownership between sequences and samples when they are attached at the beginning or end of sequencing reads. Constructing DNA barcode sets provides the candidate DNA barcodes for this application. To increase the accuracy of DNA barcode sets, a particle swarm optimization (PSO) algorithm has been modified and used to construct the DNA barcode sets in this paper. Compared with the extant results, some lower bounds of DNA barcode sets are improved. The results show that the proposed algorithm is effective in constructing DNA barcode sets.

Efficient alignment-free DNA barcode analytics

PubMed Central

Kuksa, Pavel; Pavlovic, Vladimir

2009-01-01

Background In this work we consider barcode DNA analysis problems and address them using alternative, alignment-free methods and representations which model sequences as collections of short sequence fragments (features). The methods use fixed-length representations (spectrum) for barcode sequences to measure similarities or dissimilarities between sequences coming from the same or different species. The spectrum-based representation not only allows for accurate and computationally efficient species classification, but also opens possibility for accurate clustering analysis of putative species barcodes and identification of critical within-barcode loci distinguishing barcodes of different sample groups. Results New alignment-free methods provide highly accurate and fast DNA barcode-based identification and classification of species with substantial improvements in accuracy and speed over state-of-the-art barcode analysis methods. We evaluate our methods on problems of species classification and identification using barcodes, important and relevant analytical tasks in many practical applications (adverse species movement monitoring, sampling surveys for unknown or pathogenic species identification, biodiversity assessment, etc.) On several benchmark barcode datasets, including ACG, Astraptes, Hesperiidae, Fish larvae, and Birds of North America, proposed alignment-free methods considerably improve prediction accuracy compared to prior results. We also observe significant running time improvements over the state-of-the-art methods. Conclusion Our results show that newly developed alignment-free methods for DNA barcoding can efficiently and with high accuracy identify specimens by examining only few barcode features, resulting in increased scalability and interpretability of current computational approaches to barcoding. PMID:19900305

Implementation options for DNA-based identification into ecological status assessment under the European Water Framework Directive.

PubMed

Hering, Daniel; Borja, Angel; Jones, J Iwan; Pont, Didier; Boets, Pieter; Bouchez, Agnes; Bruce, Kat; Drakare, Stina; Hänfling, Bernd; Kahlert, Maria; Leese, Florian; Meissner, Kristian; Mergen, Patricia; Reyjol, Yorick; Segurado, Pedro; Vogler, Alfried; Kelly, Martyn

2018-07-01

Assessment of ecological status for the European Water Framework Directive (WFD) is based on "Biological Quality Elements" (BQEs), namely phytoplankton, benthic flora, benthic invertebrates and fish. Morphological identification of these organisms is a time-consuming and expensive procedure. Here, we assess the options for complementing and, perhaps, replacing morphological identification with procedures using eDNA, metabarcoding or similar approaches. We rate the applicability of DNA-based identification for the individual BQEs and water categories (rivers, lakes, transitional and coastal waters) against eleven criteria, summarised under the headlines representativeness (for example suitability of current sampling methods for DNA-based identification, errors from DNA-based species detection), sensitivity (for example capability to detect sensitive taxa, unassigned reads), precision of DNA-based identification (knowledge about uncertainty), comparability with conventional approaches (for example sensitivity of metrics to differences in DNA-based identification), cost effectiveness and environmental impact. Overall, suitability of DNA-based identification is particularly high for fish, as eDNA is a well-suited sampling approach which can replace expensive and potentially harmful methods such as gill-netting, trawling or electrofishing. Furthermore, there are attempts to replace absolute by relative abundance in metric calculations. For invertebrates and phytobenthos, the main challenges include the modification of indices and completing barcode libraries. For phytoplankton, the barcode libraries are even more problematic, due to the high taxonomic diversity in plankton samples. If current assessment concepts are kept, DNA-based identification is least appropriate for macrophytes (rivers, lakes) and angiosperms/macroalgae (transitional and coastal waters), which are surveyed rather than sampled. We discuss general implications of implementing DNA-based identification into standard ecological assessment, in particular considering any adaptations to the WFD that may be required to facilitate the transition to molecular data. Copyright © 2018 Elsevier Ltd. All rights reserved.

Off the Shelf: Trends in the Purchase and Use of Electronic Reference Books

ERIC Educational Resources Information Center

Korah, Abe; Cassidy, Erin; Elmore, Eric; Jerabek, Ann

2009-01-01

What is the future direction of reference books? What types of policies are libraries implementing regarding the purchase of electronic reference books? Are libraries still buying hard copy reference items when an electronic equivalent is available? This paper discusses a national survey of libraries regarding the purchase and use of electronic…

Analysis of the Questions Asked through Digital and Face-to-Face Reference Services

ERIC Educational Resources Information Center

Tsuji, Keita; Arai, Shunsuke; Suga, Reina; Ikeuchi, Atsushi; Yoshikane, Fuyuki

2013-01-01

In Japan, only a few public libraries provide e-mail reference services. To help public libraries start e-mail reference services, the authors investigated reference questions received by libraries via e-mail and traditional face-to-face services. The authors found that research questions are more frequently observed among e-mail questions and…

User Preferences in Reference Services: Virtual Reference and Academic Libraries

ERIC Educational Resources Information Center

Cummings, Joel; Cummings, Lara; Frederiksen, Linda

2007-01-01

This study examines the use of chat in an academic library's user population and where virtual reference services might fit within the spectrum of public services offered by academic libraries. Using questionnaires, this research demonstrates that many within the academic community are open to the idea of chat-based reference or using chat for…

Browsing the Virtual Stacks: Making Electronic Reference Tools More Visible to Users

ERIC Educational Resources Information Center

Peterson, Elizabeth

2008-01-01

Electronic reference resources are expanding traditional print reference collections far beyond the walls of the library building. While the library literature has seen a debate rage about the various merits and pitfalls of electronic reference sources, no one disputes they are here to stay. As more of the library content becomes available through…

DNA Barcoding of Ichthyoplankton in Hampton Roads Bay Estuary

NASA Astrophysics Data System (ADS)

Wilkins, N.; Rodríguez, Á. E.

2016-02-01

Zooplankton is composed of animals that drift within the water column. The study of zooplankton biodiversity and distribution is crucial to understand oceanic ecosystems and anticipate the effects of climate change. In this study our focus is on ichthyoplankton (fish eggs and larvae). Our aim is to employ molecular genetic techniques such as DNA barcoding to begin a detailed characterization of ichthyoplankton diversity, abundance and community structure in the Hampton Roads Bay Estuary (HRBE). A sampling of zooplankton was performed on June 19, 2015. Samples were taken with a 0.5m, 200 µm mesh net in triplicates at two stations: inner shore in the mouth of Jones Creek and 5 miles off Hampton in the lower part of Chesapeake Bay. Physical parameters (dissolved oxygen, salinity, and temperature and water transparency) were measured simultaneously. Species were identified by DNA barcoding using the mitochondrial DNA (mtDNA) of the Cytochrome Oxidase 1 (CO1) gene. Fish eggs were identified from Opistonema oglinum (Atlantic Thread Herring) at the offshore stations while, Anchoa mitchilli was found at both stations. These species are common to the area and as observed, differences in species between stations were found. O. oglinum eggs were found in the offshore stations, which is their reported habitat. A. mitchilli eggs were found in both stations; both known to exhibit a wider salinity tolerance. This work indicates that using mtDNA-CO1 barcoding is suitable to identify ichthyoplankton to the species level and helped validate DNA barcoding as a faster taxonomic approach. The long term objective of this project is to provide taxonomic composition and biodiversity assessment of ichthyoplankton in HRBE. This data will be a reference for broad monitoring programs; for a better understanding and management of ecologically and commercially important species in the HRBE. Monthly samplings will be performed for a year beginning September 2015.

BioMaS: a modular pipeline for Bioinformatic analysis of Metagenomic AmpliconS.

PubMed

Fosso, Bruno; Santamaria, Monica; Marzano, Marinella; Alonso-Alemany, Daniel; Valiente, Gabriel; Donvito, Giacinto; Monaco, Alfonso; Notarangelo, Pasquale; Pesole, Graziano

2015-07-01

Substantial advances in microbiology, molecular evolution and biodiversity have been carried out in recent years thanks to Metagenomics, which allows to unveil the composition and functions of mixed microbial communities in any environmental niche. If the investigation is aimed only at the microbiome taxonomic structure, a target-based metagenomic approach, here also referred as Meta-barcoding, is generally applied. This approach commonly involves the selective amplification of a species-specific genetic marker (DNA meta-barcode) in the whole taxonomic range of interest and the exploration of its taxon-related variants through High-Throughput Sequencing (HTS) technologies. The accessibility to proper computational systems for the large-scale bioinformatic analysis of HTS data represents, currently, one of the major challenges in advanced Meta-barcoding projects. BioMaS (Bioinformatic analysis of Metagenomic AmpliconS) is a new bioinformatic pipeline designed to support biomolecular researchers involved in taxonomic studies of environmental microbial communities by a completely automated workflow, comprehensive of all the fundamental steps, from raw sequence data upload and cleaning to final taxonomic identification, that are absolutely required in an appropriately designed Meta-barcoding HTS-based experiment. In its current version, BioMaS allows the analysis of both bacterial and fungal environments starting directly from the raw sequencing data from either Roche 454 or Illumina HTS platforms, following two alternative paths, respectively. BioMaS is implemented into a public web service available at https://recasgateway.ba.infn.it/ and is also available in Galaxy at http://galaxy.cloud.ba.infn.it:8080 (only for Illumina data). BioMaS is a friendly pipeline for Meta-barcoding HTS data analysis specifically designed for users without particular computing skills. A comparative benchmark, carried out by using a simulated dataset suitably designed to broadly represent the currently known bacterial and fungal world, showed that BioMaS outperforms QIIME and MOTHUR in terms of extent and accuracy of deep taxonomic sequence assignments.

The real maccoyii: identifying tuna sushi with DNA barcodes--contrasting characteristic attributes and genetic distances.

PubMed

Lowenstein, Jacob H; Amato, George; Kolokotronis, Sergios-Orestis

2009-11-18

The use of DNA barcodes for the identification of described species is one of the least controversial and most promising applications of barcoding. There is no consensus, however, as to what constitutes an appropriate identification standard and most barcoding efforts simply attempt to pair a query sequence with reference sequences and deem identification successful if it falls within the bounds of some pre-established cutoffs using genetic distance. Since the Renaissance, however, most biological classification schemes have relied on the use of diagnostic characters to identify and place species. Here we developed a cytochrome c oxidase subunit I character-based key for the identification of all tuna species of the genus Thunnus, and compared its performance with distance-based measures for identification of 68 samples of tuna sushi purchased from 31 restaurants in Manhattan (New York City) and Denver, Colorado. Both the character-based key and GenBank BLAST successfully identified 100% of the tuna samples, while the Barcode of Life Database (BOLD) as well as genetic distance thresholds, and neighbor-joining phylogenetic tree building performed poorly in terms of species identification. A piece of tuna sushi has the potential to be an endangered species, a fraud, or a health hazard. All three of these cases were uncovered in this study. Nineteen restaurant establishments were unable to clarify or misrepresented what species they sold. Five out of nine samples sold as a variant of "white tuna" were not albacore (T. alalunga), but escolar (Lepidocybium flavorunneum), a gempylid species banned for sale in Italy and Japan due to health concerns. Nineteen samples were northern bluefin tuna (T. thynnus) or the critically endangered southern bluefin tuna (T. maccoyii), though nine restaurants that sold these species did not state these species on their menus. The Convention on International Trade Endangered Species (CITES) requires that listed species must be identifiable in trade. This research fulfills this requirement for tuna, and supports the nomination of northern bluefin tuna for CITES listing in 2010.

Feasibility and Limitations of Vaccine Two-Dimensional Barcoding Using Mobile Devices.

PubMed

Bell, Cameron; Guerinet, Julien; Atkinson, Katherine M; Wilson, Kumanan

2016-06-23

Two-dimensional (2D) barcoding has the potential to enhance documentation of vaccine encounters at the point of care. However, this is currently limited to environments equipped with dedicated barcode scanners and compatible record systems. Mobile devices may present a cost-effective alternative to leverage 2D vaccine vial barcodes and improve vaccine product-specific information residing in digital health records. Mobile devices have the potential to capture product-specific information from 2D vaccine vial barcodes. We sought to examine the feasibility, performance, and potential limitations of scanning 2D barcodes on vaccine vials using 4 different mobile phones. A unique barcode scanning app was developed for Android and iOS operating systems. The impact of 4 variables on the scan success rate, data accuracy, and time to scan were examined: barcode size, curvature, fading, and ambient lighting conditions. Two experimenters performed 4 trials 10 times each, amounting to a total of 2160 barcode scan attempts. Of the 1832 successful scans performed in this evaluation, zero produced incorrect data. Five-millimeter barcodes were the slowest to scan, although only by 0.5 seconds on average. Barcodes with up to 50% fading had a 100% success rate, but success rate deteriorated beyond 60% fading. Curved barcodes took longer to scan compared with flat, but success rate deterioration was only observed at a vial diameter of 10 mm. Light conditions did not affect success rate or scan time between 500 lux and 20 lux. Conditions below 20 lux impeded the device's ability to scan successfully. Variability in scan time was observed across devices in all trials performed. 2D vaccine barcoding is possible using mobile devices and is successful under the majority of conditions examined. Manufacturers utilizing 2D barcodes should take into consideration the impact of factors that limit scan success rates. Future studies should evaluate the effect of mobile barcoding on workflow and vaccine administrator acceptance.

Newborn Screening: MedlinePlus Health Topic

MedlinePlus

... deficiency (National Library of Medicine) Genetics Home Reference: glutaric acidemia type I (National Library of Medicine) Genetics Home Reference: glutaric acidemia type II (National Library of Medicine) Genetics ...

A DNA mini-barcode for land plants.

PubMed

Little, Damon P

2014-05-01

Small portions of the barcode region - mini-barcodes - may be used in place of full-length barcodes to overcome DNA degradation for samples with poor DNA preservation. 591,491,286 rbcL mini-barcode primer combinations were electronically evaluated for PCR universality, and two novel highly universal sets of priming sites were identified. Novel and published rbcL mini-barcode primers were evaluated for PCR amplification [determined with a validated electronic simulation (n = 2765) and empirically (n = 188)], Sanger sequence quality [determined empirically (n = 188)], and taxonomic discrimination [determined empirically (n = 30,472)]. PCR amplification for all mini-barcodes, as estimated by validated electronic simulation, was successful for 90.2-99.8% of species. Overall Sanger sequence quality for mini-barcodes was very low - the best mini-barcode tested produced sequences of adequate quality (B20 ≥ 0.5) for 74.5% of samples. The majority of mini-barcodes provide correct identifications of families in excess of 70.1% of the time. Discriminatory power noticeably decreased at lower taxonomic levels. At the species level, the discriminatory power of the best mini-barcode was less than 38.2%. For samples believed to contain DNA from only one species, an investigator should attempt to sequence, in decreasing order of utility and probability of success, mini-barcodes F (rbcL1/rbcLB), D (F52/R193) and K (F517/R604). For samples believed to contain DNA from more than one species, an investigator should amplify and sequence mini-barcode D (F52/R193). © 2013 John Wiley & Sons Ltd.

Virtual Reference Transcript Analysis: A Few Models.

ERIC Educational Resources Information Center

Smyth, Joanne

2003-01-01

Describes the introduction of virtual, or digital, reference service at the University of New Brunswick libraries. Highlights include analyzing transcripts from LIVE (Library Information in a Virtual Environment); reference question types; ACRL (Association of College and Research Libraries) information literacy competency standards; and the Big 6…

Reference Desk Is Not Dead Yet: A Perspective from the National Medical Library of Cuba

ERIC Educational Resources Information Center

Arroyo, Sonia Santana

2015-01-01

There persists an intense debate on whether or not the traditional reference desk should be in academic libraries. Yet, despite many anti-desk studies, the place of the reference desk still remains. This paper aims to review the current significance of the reference desk for some libraries, as well as the importance of choosing the proper…

A DNA Mini-Barcoding System for Authentication of Processed Fish Products.

PubMed

Shokralla, Shadi; Hellberg, Rosalee S; Handy, Sara M; King, Ian; Hajibabaei, Mehrdad

2015-10-30

Species substitution is a form of seafood fraud for the purpose of economic gain. DNA barcoding utilizes species-specific DNA sequence information for specimen identification. Previous work has established the usability of short DNA sequences-mini-barcodes-for identification of specimens harboring degraded DNA. This study aims at establishing a DNA mini-barcoding system for all fish species commonly used in processed fish products in North America. Six mini-barcode primer pairs targeting short (127-314 bp) fragments of the cytochrome c oxidase I (CO1) DNA barcode region were developed by examining over 8,000 DNA barcodes from species in the U.S. Food and Drug Administration (FDA) Seafood List. The mini-barcode primer pairs were then tested against 44 processed fish products representing a range of species and product types. Of the 44 products, 41 (93.2%) could be identified at the species or genus level. The greatest mini-barcoding success rate found with an individual primer pair was 88.6% compared to 20.5% success rate achieved by the full-length DNA barcode primers. Overall, this study presents a mini-barcoding system that can be used to identify a wide range of fish species in commercial products and may be utilized in high throughput DNA sequencing for authentication of heavily processed fish products.

Four years of DNA barcoding: current advances and prospects.

PubMed

Frézal, Lise; Leblois, Raphael

2008-09-01

Research using cytochrome c oxidase barcoding techniques on zoological specimens was initiated by Hebert et al. [Hebert, P.D.N., Ratnasingham, S., deWaard, J.R., 2003. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proc. R. Soc. Lond. B 270, S96-S99]. By March 2004, the Consortium for the Barcode of Life started to promote the use of a standardized DNA barcoding approach, consisting of identifying a specimen as belonging to a certain animal species based on a single universal marker: the DNA barcode sequence. Over the last 4 years, this approach has become increasingly popular and advances as well as limitations have clearly emerged as increasing amounts of organisms have been studied. Our purpose is to briefly expose DNA Barcode of Life principles, pros and cons, relevance and universality. The initially proposed Barcode of life framework has greatly evolved, giving rise to a flexible description of DNA barcoding and a larger range of applications.

Exploring the utility of DNA barcoding in species delimitation of Polypedilum (Tripodura) non-biting midges (Diptera: Chironomidae).

PubMed

Song, Chao; Wang, Qian; Zhang, Ruilei; Sun, Bingjiao; Wang, Xinhua

2016-02-16

In this study, we tested the utility of the mitochondrial gene cytochrome c oxidase subunit 1 (CO1) as the barcode region to deal with taxonomical problems of Polypedilum (Tripodura) non-biting midges (Diptera: Chironomidae). The 114 DNA barcodes representing 27 morphospecies are divided into 33 well separated clusters based on both Neighbor Joining and Maximum Likelihood methods. DNA barcodes revealed an 82% success rate in matching with morphospecies. The selected DNA barcode data support 37-64 operational taxonomic units (OTUs) based on the methods of Automatic Barcode Gap Discovery (ABGD) and Poisson Tree Process (PTP). Furthermore, a priori species based on consistent phenotypic variations were attested by molecular analysis, and a taxonomical misidentification of barcode sequences from GenBank was found. We could not observe a distinct barcode gap but an overlap ranged from 9-12%. Our results supported DNA barcoding as an ideal method to detect cryptic species, delimit sibling species, and associate different life stages in non-biting midges.

DNA barcoding in the media: does coverage of cool science reflect its social context?

PubMed

Geary, Janis; Camicioli, Emma; Bubela, Tania

2016-09-01

Paul Hebert and colleagues first described DNA barcoding in 2003, which led to international efforts to promote and coordinate its use. Since its inception, DNA barcoding has generated considerable media coverage. We analysed whether this coverage reflected both the scientific and social mandates of international barcoding organizations. We searched newspaper databases to identify 900 English-language articles from 2003 to 2013. Coverage of the science of DNA barcoding was highly positive but lacked context for key topics. Coverage omissions pose challenges for public understanding of the science and applications of DNA barcoding; these included coverage of governance structures and issues related to the sharing of genetic resources across national borders. Our analysis provided insight into how barcoding communication efforts have translated into media coverage; more targeted communication efforts may focus media attention on previously omitted, but important topics. Our analysis is timely as the DNA barcoding community works to establish the International Society for the Barcode of Life.

75 FR 56922 - Implementation of the Intelligent Mail Package Barcode

Federal Register 2010, 2011, 2012, 2013, 2014

2010-09-17

... the USPS Intelligent Mail strategy. Packages that currently bear barcodes designed to provide delivery... symbology of the barcode; however the elements within the barcode and layout will change. There are several...

BioBarcode: a general DNA barcoding database and server platform for Asian biodiversity resources.

PubMed

Lim, Jeongheui; Kim, Sang-Yoon; Kim, Sungmin; Eo, Hae-Seok; Kim, Chang-Bae; Paek, Woon Kee; Kim, Won; Bhak, Jong

2009-12-03

DNA barcoding provides a rapid, accurate, and standardized method for species-level identification using short DNA sequences. Such a standardized identification method is useful for mapping all the species on Earth, particularly when DNA sequencing technology is cheaply available. There are many nations in Asia with many biodiversity resources that need to be mapped and registered in databases. We have built a general DNA barcode data processing system, BioBarcode, with open source software - which is a general purpose database and server. It uses mySQL RDBMS 5.0, BLAST2, and Apache httpd server. An exemplary database of BioBarcode has around 11,300 specimen entries (including GenBank data) and registers the biological species to map their genetic relationships. The BioBarcode database contains a chromatogram viewer which improves the performance in DNA sequence analyses. Asia has a very high degree of biodiversity and the BioBarcode database server system aims to provide an efficient bioinformatics protocol that can be freely used by Asian researchers and research organizations interested in DNA barcoding. The BioBarcode promotes the rapid acquisition of biological species DNA sequence data that meet global standards by providing specialized services, and provides useful tools that will make barcoding cheaper and faster in the biodiversity community such as standardization, depository, management, and analysis of DNA barcode data. The system can be downloaded upon request, and an exemplary server has been constructed with which to build an Asian biodiversity system http://www.asianbarcode.org.

Reliable DNA Barcoding Performance Proved for Species and Island Populations of Comoran Squamate Reptiles

PubMed Central

Hawlitschek, Oliver; Nagy, Zoltán T.; Berger, Johannes; Glaw, Frank

2013-01-01

In the past decade, DNA barcoding became increasingly common as a method for species identification in biodiversity inventories and related studies. However, mainly due to technical obstacles, squamate reptiles have been the target of few barcoding studies. In this article, we present the results of a DNA barcoding study of squamates of the Comoros archipelago, a poorly studied group of oceanic islands close to and mostly colonized from Madagascar. The barcoding dataset presented here includes 27 of the 29 currently recognized squamate species of the Comoros, including 17 of the 18 endemic species. Some species considered endemic to the Comoros according to current taxonomy were found to cluster with non-Comoran lineages, probably due to poorly resolved taxonomy. All other species for which more than one barcode was obtained corresponded to distinct clusters useful for species identification by barcoding. In most species, even island populations could be distinguished using barcoding. Two cryptic species were identified using the DNA barcoding approach. The obtained barcoding topology, a Bayesian tree based on COI sequences of 5 genera, was compared with available multigene topologies, and in 3 cases, major incongruences between the two topologies became evident. Three of the multigene studies were initiated after initial screening of a preliminary version of the barcoding dataset presented here. We conclude that in the case of the squamates of the Comoros Islands, DNA barcoding has proven a very useful and efficient way of detecting isolated populations and promising starting points for subsequent research. PMID:24069192

Reference and Bibliographic Instruction: A Survey of Philosophy Statements in LIBRAS Libraries.

ERIC Educational Resources Information Center

Keith, Ellen; Kohut, Dave

1998-01-01

A survey of statements of philosophy on reference and bibliographic instruction from libraries in small, Chicago area liberal arts colleges and universities revealed a wide variety of relationships between the two services. The study concluded that most of the libraries surveyed compartmentalized reference and bibliographic instruction. (PEN)

An Integrated RFID and Barcode Tagged Item Inventory System for Deployment at New Brunswick Laboratory

DOE Office of Scientific and Technical Information (OSTI.GOV)

Younkin, James R; Kuhn, Michael J; Gradle, Colleen

New Brunswick Laboratory (NBL) has a numerous inventory containing thousands of plutonium and uranium certified reference materials. The current manual inventory process is well established but is a lengthy process which requires significant oversight and double checking to ensure correctness. Oak Ridge National Laboratory has worked with NBL to develop and deploy a new inventory system which utilizes handheld computers with barcode scanners and radio frequency identification (RFID) readers termed the Tagged Item Inventory System (TIIS). Certified reference materials are identified by labels which incorporate RFID tags and barcodes. The label printing process and RFID tag association process are integratedmore » into the main desktop software application. Software on the handheld computers syncs with software on designated desktop machines and the NBL inventory database to provide a seamless inventory process. This process includes: 1) identifying items to be inventoried, 2) downloading the current inventory information to the handheld computer, 3) using the handheld to read item and location labels, and 4) syncing the handheld computer with a designated desktop machine to analyze the results, print reports, etc. The security of this inventory software has been a major concern. Designated roles linked to authenticated logins are used to control access to the desktop software while password protection and badge verification are used to control access to the handheld computers. The overall system design and deployment at NBL will be presented. The performance of the system will also be discussed with respect to a small piece of the overall inventory. Future work includes performing a full inventory at NBL with the Tagged Item Inventory System and comparing performance, cost, and radiation exposures to the current manual inventory process.« less

DNA Barcoding Coupled with High Resolution Melting Analysis Enables Rapid and Accurate Distinction of Aspergillus species.

PubMed

Fidler, Gabor; Kocsube, Sandor; Leiter, Eva; Biro, Sandor; Paholcsek, Melinda

2017-08-01

We describe a high-resolution melting (HRM) analysis method that is rapid, reproducible, and able to identify reference strains and further 40 clinical isolates of Aspergillus fumigatus (14), A. lentulus (3), A. terreus (7), A. flavus (8), A. niger (2), A. welwitschiae (4), and A. tubingensis (2). Asp1 and Asp2 primer sets were designed to amplify partial sequences of the Aspergillus benA (beta-tubulin) genes in a closed-, single-tube system. Human placenta DNA, further Aspergillus (3), Candida (9), Fusarium (6), and Scedosporium (2) nucleic acids from type strains and clinical isolates were also included in this study to evaluate cross reactivity with other relevant pathogens causing invasive fungal infections. The barcoding capacity of this method proved to be 100% providing distinctive binomial scores; 14, 34, 36, 35, 25, 15, 26 when tested among species, while the within-species distinction capacity of the assay proved to be 0% based on the aligned thermodynamic profiles of the Asp1, Asp2 melting clusters allowing accurate species delimitation of all tested clinical isolates. The identification limit of this HRM assay was also estimated on Aspergillus reference gDNA panels where it proved to be 10-102 genomic equivalents (GE) except the A. fumigatus panel where it was 103 only. Furthermore, misidentification was not detected with human genomic DNA or with Candida, Fusarium, and Scedosporium strains. Our DNA barcoding assay introduced here provides results within a few hours, and it may possess further diagnostic utility when analyzing standard cultures supporting adequate therapeutic decisions. © The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Watermarking spot colors in packaging

NASA Astrophysics Data System (ADS)

Reed, Alastair; Filler, TomáÅ.¡; Falkenstern, Kristyn; Bai, Yang

2015-03-01

In January 2014, Digimarc announced Digimarc® Barcode for the packaging industry to improve the check-out efficiency and customer experience for retailers. Digimarc Barcode is a machine readable code that carries the same information as a traditional Universal Product Code (UPC) and is introduced by adding a robust digital watermark to the package design. It is imperceptible to the human eye but can be read by a modern barcode scanner at the Point of Sale (POS) station. Compared to a traditional linear barcode, Digimarc Barcode covers the whole package with minimal impact on the graphic design. This significantly improves the Items per Minute (IPM) metric, which retailers use to track the checkout efficiency since it closely relates to their profitability. Increasing IPM by a few percent could lead to potential savings of millions of dollars for retailers, giving them a strong incentive to add the Digimarc Barcode to their packages. Testing performed by Digimarc showed increases in IPM of at least 33% using the Digimarc Barcode, compared to using a traditional barcode. A method of watermarking print ready image data used in the commercial packaging industry is described. A significant proportion of packages are printed using spot colors, therefore spot colors needs to be supported by an embedder for Digimarc Barcode. Digimarc Barcode supports the PANTONE spot color system, which is commonly used in the packaging industry. The Digimarc Barcode embedder allows a user to insert the UPC code in an image while minimizing perceptibility to the Human Visual System (HVS). The Digimarc Barcode is inserted in the printing ink domain, using an Adobe Photoshop plug-in as the last step before printing. Since Photoshop is an industry standard widely used by pre-press shops in the packaging industry, a Digimarc Barcode can be easily inserted and proofed.

Field calibration of blowfly-derived DNA against traditional methods for assessing mammal diversity in tropical forests.

PubMed

Lee, Ping-Shin; Gan, Han Ming; Clements, Gopalasamy Reuben; Wilson, John-James

2016-11-01

Mammal diversity assessments based on DNA derived from invertebrates have been suggested as alternatives to assessments based on traditional methods; however, no study has field-tested both approaches simultaneously. In Peninsular Malaysia, we calibrated the performance of mammal DNA derived from blowflies (Diptera: Calliphoridae) against traditional methods used to detect species. We first compared five methods (cage trapping, mist netting, hair trapping, scat collection, and blowfly-derived DNA) in a forest reserve with no recent reports of megafauna. Blowfly-derived DNA and mist netting detected the joint highest number of species (n = 6). Only one species was detected by multiple methods. Compared to the other methods, blowfly-derived DNA detected both volant and non-volant species. In another forest reserve, rich in megafauna, we calibrated blowfly-derived DNA against camera traps. Blowfly-derived DNA detected more species (n = 11) than camera traps (n = 9), with only one species detected by both methods. The rarefaction curve indicated that blowfly-derived DNA would continue to detect more species with greater sampling effort. With further calibration, blowfly-derived DNA may join the list of traditional field methods. Areas for further investigation include blowfly feeding and dispersal biology, primer biases, and the assembly of a comprehensive and taxonomically-consistent DNA barcode reference library.

Stranded Whole Transcriptome RNA-Seq for All RNA Types

PubMed Central

Yan, Pearlly X.; Fang, Fang; Buechlein, Aaron; Ford, James B.; Tang, Haixu; Huang, Tim H.; Burow, Matthew E.; Liu, Yunlong; Rusch, Douglas B.

2015-01-01

Stranded whole transcriptome RNA-Seq described in this unit captures quantitative expression data for all types of RNA including, but not limited to miRNA (microRNA), piRNA (Piwi-interacting RNA), snoRNA (small nucleolar RNA), lincRNA (large non-coding intergenic RNA), SRP RNA (signal recognition particle RNA), tRNA (transfer RNA), mtRNA (mitochondrial RNA) and mRNA (messenger RNA). The size and nature of these types of RNA are irrelevant to the approach described here. Barcoded libraries for multiplexing on the Illumina platform are generated with this approach but it can be applied to other platforms with a few modifications. PMID:25599667

DNA barcoding of invasive plants in China: A resource for identifying invasive plants.

PubMed

Xu, Song-Zhi; Li, Zhen-Yu; Jin, Xiao-Hua

2018-01-01

Invasive plants have aroused attention globally for causing ecological damage and having a negative impact on the economy and human health. However, it can be extremely challenging to rapidly and accurately identify invasive plants based on morphology because they are an assemblage of many different families and many plant materials lack sufficient diagnostic characteristics during border inspections. It is therefore urgent to evaluate candidate loci and build a reliable genetic library to prevent invasive plants from entering China. In this study, five common single markers (ITS, ITS2, matK, rbcL and trnH-psbA) were evaluated using 634 species (including 469 invasive plant species in China, 10 new records to China, 16 potentially invasive plant species around the world but not introduced into China yet and 139 plant species native to China) based on three different methods. Our results indicated that ITS2 displayed largest intra- and interspecific divergence (1.72% and 91.46%). Based on NJ tree method, ITS2, ITS, matK, rbcL and trnH-psbA provided 76.84%, 76.5%, 63.21%, 52.86% and 50.68% discrimination rates, respectively. The combination of ITS + matK performed best and provided 91.03% discriminatory power, followed by ITS2 + matK (85.78%). For identifying unknown individuals, ITS + matK had 100% correct identification rate based on our database, followed by ITS/ITS2 (both 93.33%) and ITS2 + matK (91.67%). Thus, we propose ITS/ITS2 + matK as the most suitable barcode for invasive plants in China. This study also demonstrated that DNA barcoding is an efficient tool for identifying invasive species. © 2017 John Wiley & Sons Ltd.

[Trial of eye drops recognizer for visually disabled persons].

PubMed

Okamoto, Norio; Suzuki, Katsuhiko; Mimura, Osamu

2009-01-01

The development of a device to enable the visually disabled to differentiate eye drops and their dose. The new instrument is composed of a voice generator and a two-dimensional bar-code reader (LS9208). We designed voice outputs for the visually disabled to state when (number of times) and where (right, left, or both) to administer eye drops. We then determined the minimum bar-code size that can be recognized. After attaching bar-codes of the appropriate size to the lateral or bottom surface of the eye drops container, the readability of the bar-codes was compared. The minimum discrimination bar-code size was 6 mm high x 8.5 mm long. Bar-codes on the bottom surface could be more easily recognized than bar-codes on the side. Our newly-developed device using bar-codes enables visually disabled persons to differentiate eye drops and their doses.

Detection of dopamine in dopaminergic cell using nanoparticles-based barcode DNA analysis.

PubMed

An, Jeung Hee; Kim, Tae-Hyung; Oh, Byung-Keun; Choi, Jeong Woo

2012-01-01

Nanotechnology-based bio-barcode-amplification analysis may be an innovative approach to dopamine detection. In this study, we evaluated the efficacy of this bio-barcode DNA method in detecting dopamine from dopaminergic cells. Herein, a combination DNA barcode and bead-based immunoassay for neurotransmitter detection with PCR-like sensitivity is described. This method relies on magnetic nanoparticles with antibodies and nanoparticles that are encoded with DNA, and antibodies that can sandwich the target protein captured by the nanoparticle-bound antibodies. The aggregate sandwich structures are magnetically separated from solution, and treated in order to remove the conjugated barcode DNA. The DNA barcodes were then identified via PCR analysis. The dopamine concentration in dopaminergic cells can be readily and rapidly detected via the bio-barcode assay method. The bio-barcode assay method is, therefore, a rapid and high-throughput screening tool for the detection of neurotransmitters such as dopamine.

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing.

PubMed

Stiffler, Michael A; Subramanian, Subu K; Salinas, Victor H; Ranganathan, Rama

2016-07-03

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 β-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to β-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

TagDust2: a generic method to extract reads from sequencing data.

PubMed

Lassmann, Timo

2015-01-28

Arguably the most basic step in the analysis of next generation sequencing data (NGS) involves the extraction of mappable reads from the raw reads produced by sequencing instruments. The presence of barcodes, adaptors and artifacts subject to sequencing errors makes this step non-trivial. Here I present TagDust2, a generic approach utilizing a library of hidden Markov models (HMM) to accurately extract reads from a wide array of possible read architectures. TagDust2 extracts more reads of higher quality compared to other approaches. Processing of multiplexed single, paired end and libraries containing unique molecular identifiers is fully supported. Two additional post processing steps are included to exclude known contaminants and filter out low complexity sequences. Finally, TagDust2 can automatically detect the library type of sequenced data from a predefined selection. Taken together TagDust2 is a feature rich, flexible and adaptive solution to go from raw to mappable NGS reads in a single step. The ability to recognize and record the contents of raw reads will help to automate and demystify the initial, and often poorly documented, steps in NGS data analysis pipelines. TagDust2 is freely available at: http://tagdust.sourceforge.net .

The complete genome sequence of a third distinct baculovirus isolated from the true armyworm, Mythimna unipuncta, contains two copies of the lef-7 gene

USDA-ARS?s Scientific Manuscript database

A baculovirus isolate from a USDA Forest Service collection was examined by electron microscopy and analysis of its genome sequence. The isolate, formerly referred to as Pseudoletia (Mythimna) sp. nucleopolyhedrovirus #7 (MyspNPV#7), was determined by barcoding PCR to derive from the host species My...

Learning to Provide 3D Virtual Reference: A Library Science Assignment

ERIC Educational Resources Information Center

Johnson, Megan; Purpur, Geraldine; Abbott, Lisa T.

2009-01-01

In spring semester 2009, two of the authors taught LIB 5020--Information Sources & Services to graduate library science students at Appalachian State University. The course covers information seeking patterns and provides an overview of reference services. The course is also designed to examine and evaluate library reference materials and…

Virtual Reference Services--Down-Under: A Cautionary Tale.

ERIC Educational Resources Information Center

Wagner, Gulten S.

Digital reference services at university libraries in Australia and New Zealand are a recent phenomena dating back to the late 1990s--following the developments in Web-based online library services. This paper examines the move towards the provision of e-mail reference services based on the study of 16 randomly chosen university libraries in…

BioBarcode: a general DNA barcoding database and server platform for Asian biodiversity resources

PubMed Central

2009-01-01

Background DNA barcoding provides a rapid, accurate, and standardized method for species-level identification using short DNA sequences. Such a standardized identification method is useful for mapping all the species on Earth, particularly when DNA sequencing technology is cheaply available. There are many nations in Asia with many biodiversity resources that need to be mapped and registered in databases. Results We have built a general DNA barcode data processing system, BioBarcode, with open source software - which is a general purpose database and server. It uses mySQL RDBMS 5.0, BLAST2, and Apache httpd server. An exemplary database of BioBarcode has around 11,300 specimen entries (including GenBank data) and registers the biological species to map their genetic relationships. The BioBarcode database contains a chromatogram viewer which improves the performance in DNA sequence analyses. Conclusion Asia has a very high degree of biodiversity and the BioBarcode database server system aims to provide an efficient bioinformatics protocol that can be freely used by Asian researchers and research organizations interested in DNA barcoding. The BioBarcode promotes the rapid acquisition of biological species DNA sequence data that meet global standards by providing specialized services, and provides useful tools that will make barcoding cheaper and faster in the biodiversity community such as standardization, depository, management, and analysis of DNA barcode data. The system can be downloaded upon request, and an exemplary server has been constructed with which to build an Asian biodiversity system http://www.asianbarcode.org. PMID:19958506

Pay Attention to the Overlooked Cryptic Diversity in Existing Barcoding Data: the Case of Mollusca with Character-Based DNA Barcoding.

PubMed

Zou, Shanmei; Li, Qi

2016-06-01

With the global biodiversity crisis, DNA barcoding aims for fast species identification and cryptic species diversity revelation. For more than 10 years, large amounts of DNA barcode data have been accumulating in publicly available databases, most of which were conducted by distance or tree-building methods that have often been argued, especially for cryptic species revelation. In this context, overlooked cryptic diversity may exist in the available barcoding data. The character-based DNA barcoding, however, has a good chance for detecting the overlooked cryptic diversity. In this study, marine mollusk was as the ideal case for detecting the overlooked potential cryptic species from existing cytochrome c oxidase I (COI) sequences with character-based DNA barcode. A total of 1081 COI sequences of mollusks, belonging to 176 species of 25 families of Gastropoda, Cephalopoda, and Lamellibranchia, were conducted by character analysis. As a whole, the character-based barcoding results were consistent with previous distance and tree-building analysis for species discrimination. More importantly, quite a number of species analyzed were divided into distinct clades with unique diagnostical characters. Based on the concept of cryptic species revelation of character-based barcoding, these species divided into separate taxonomic groups might be potential cryptic species. The detection of the overlooked potential cryptic diversity proves that the character-based barcoding mode possesses more advantages of revealing cryptic biodiversity. With the development of DNA barcoding, making the best use of barcoding data is worthy of our attention for species conservation.

Evaluation of candidate barcoding markers in Orinus (Poaceae).

PubMed

Su, X; Liu, Y P; Chen, Z; Chen, K L

2016-04-26

Orinus is an alpine endemic genus of Poaceae. Because of the imperfect specimens, high level of intraspecific morphological variability, and homoplasies of morphological characters, it is relatively difficult to delimitate species of Orinus by using morphology alone. To this end, the DNA barcoding has shown great potential in identifying species. The present study is the first attempt to test the feasibility of four proposed DNA barcoding markers (matK, rbcL, trnH-psbA, and ITS) in identifying four currently revised species of Orinus from the Qinghai-Tibetan Plateau (QTP). Among all the single-barcode candidates, the differentiation power was the highest for the nuclear internal transcribed spacer (ITS), while the chloroplast barcodes matK (M), rbcL (R), and trnH-psbA (H) could not identify the species. Meanwhile, the differentiation efficiency of the nuclear ITS (I) was also higher than any two- or three-locus combination of chloroplast barcodes, or even a combination of ITS and any chloroplast barcode except H + I and R + I. All the combinations of chloroplast barcodes plus the nuclear ITS, H + I, and R + I differentiated the highest portion of species. The highest differentiation rate for the barcodes or barcode combinations examined here was 100% (H + I and R + I). In summary, this case study showed that the nuclear ITS region represents a more promising barcode than any maternally inherited chloroplast region or combination of chloroplast regions in differentiating Orinus species from the QTP. Moreover, combining the ITS region with chloroplast regions may improve the barcoding success rate.

Data Release: DNA barcodes of plant species collected for the Global Genome Initiative for Gardens Program, National Museum of Natural History, Smithsonian Institution

PubMed Central

Zúñiga, Jose D.; Gostel, Morgan R.; Mulcahy, Daniel G.; Barker, Katharine; Asia Hill; Sedaghatpour, Maryam; Vo, Samantha Q.; Funk, Vicki A.; Coddington, Jonathan A.

2017-01-01

Abstract The Global Genome Initiative has sequenced and released 1961 DNA barcodes for genetic samples obtained as part of the Global Genome Initiative for Gardens Program. The dataset includes barcodes for 29 plant families and 309 genera that did not have sequences flagged as barcodes in GenBank and sequences from officially recognized barcoding genetic markers meet the data standard of the Consortium for the Barcode of Life. The genetic samples were deposited in the Smithsonian Institution’s National Museum of Natural History Biorepository and their records were made public through the Global Genome Biodiversity Network’s portal. The DNA barcodes are now available on GenBank. PMID:29118648

Library and Information Networks: Centralization and Decentralization.

ERIC Educational Resources Information Center

Segal, JoAnn S.

1988-01-01

Describes the development of centralized library networks and the current factors that make library sharing on a smaller scale feasible. The discussion covers the need to decide the level at which library cooperation should occur and the possibility of linking via the Open System Interface Reference Model. (37 references) (CLB)

DNA barcoding the floras of biodiversity hotspots.

PubMed

Lahaye, Renaud; van der Bank, Michelle; Bogarin, Diego; Warner, Jorge; Pupulin, Franco; Gigot, Guillaume; Maurin, Olivier; Duthoit, Sylvie; Barraclough, Timothy G; Savolainen, Vincent

2008-02-26

DNA barcoding is a technique in which species identification is performed by using DNA sequences from a small fragment of the genome, with the aim of contributing to a wide range of ecological and conservation studies in which traditional taxonomic identification is not practical. DNA barcoding is well established in animals, but there is not yet any universally accepted barcode for plants. Here, we undertook intensive field collections in two biodiversity hotspots (Mesoamerica and southern Africa). Using >1,600 samples, we compared eight potential barcodes. Going beyond previous plant studies, we assessed to what extent a "DNA barcoding gap" is present between intra- and interspecific variations, using multiple accessions per species. Given its adequate rate of variation, easy amplification, and alignment, we identified a portion of the plastid matK gene as a universal DNA barcode for flowering plants. Critically, we further demonstrate the applicability of DNA barcoding for biodiversity inventories. In addition, analyzing >1,000 species of Mesoamerican orchids, DNA barcoding with matK alone reveals cryptic species and proves useful in identifying species listed in Convention on International Trade of Endangered Species (CITES) appendixes.

DNA barcoding the floras of biodiversity hotspots

PubMed Central

Lahaye, Renaud; van der Bank, Michelle; Bogarin, Diego; Warner, Jorge; Pupulin, Franco; Gigot, Guillaume; Maurin, Olivier; Duthoit, Sylvie; Barraclough, Timothy G.; Savolainen, Vincent

2008-01-01

DNA barcoding is a technique in which species identification is performed by using DNA sequences from a small fragment of the genome, with the aim of contributing to a wide range of ecological and conservation studies in which traditional taxonomic identification is not practical. DNA barcoding is well established in animals, but there is not yet any universally accepted barcode for plants. Here, we undertook intensive field collections in two biodiversity hotspots (Mesoamerica and southern Africa). Using >1,600 samples, we compared eight potential barcodes. Going beyond previous plant studies, we assessed to what extent a “DNA barcoding gap” is present between intra- and interspecific variations, using multiple accessions per species. Given its adequate rate of variation, easy amplification, and alignment, we identified a portion of the plastid matK gene as a universal DNA barcode for flowering plants. Critically, we further demonstrate the applicability of DNA barcoding for biodiversity inventories. In addition, analyzing >1,000 species of Mesoamerican orchids, DNA barcoding with matK alone reveals cryptic species and proves useful in identifying species listed in Convention on International Trade of Endangered Species (CITES) appendixes. PMID:18258745

Tamper-indicating barcode and method

DOEpatents

Cummings, Eric B.; Even, Jr., William R.; Simmons, Blake A.; Dentinger, Paul Michael

2005-03-22

A novel tamper-indicating barcode methodology is disclosed that allows for detection of alteration to the barcode. The tamper-indicating methodology makes use of a tamper-indicating means that may be comprised of a particulate indicator, an optical indicator, a deformable substrate, and/or may be an integrated aspect of the barcode itself. This tamper-indicating information provides greater security for the contents of containers sealed with the tamper-indicating barcodes.

DNA barcoding reveals a cryptic nemertean invasion in Atlantic and Mediterranean waters

NASA Astrophysics Data System (ADS)

Fernández-Álvarez, Fernando Ángel; Machordom, Annie

2013-09-01

For several groups, like nemerteans, morphology-based identification is a hard discipline, but DNA barcoding may help non-experts in the identification process. In this study, DNA barcoding is used to reveal the cryptic invasion of Pacific Cephalothrix cf. simula into Atlantic and Mediterranean coasts. Although DNA barcoding is a promising method for the identification of Nemertea, only 6 % of the known number of nemertean species is currently associated with a correct DNA barcode. Therefore, additional morphological and molecular studies are necessary to advance the utility of DNA barcoding in the characterisation of possible nemertean alien invasions.

Evaluation of the DNA barcodes in Dendrobium (Orchidaceae) from mainland Asia.

PubMed

Xu, Songzhi; Li, Dezhu; Li, Jianwu; Xiang, Xiaoguo; Jin, Weitao; Huang, Weichang; Jin, Xiaohua; Huang, Luqi

2015-01-01

DNA barcoding has been proposed to be one of the most promising tools for accurate and rapid identification of taxa. However, few publications have evaluated the efficiency of DNA barcoding for the large genera of flowering plants. Dendrobium, one of the largest genera of flowering plants, contains many species that are important in horticulture, medicine and biodiversity conservation. Besides, Dendrobium is a notoriously difficult group to identify. DNA barcoding was expected to be a supplementary means for species identification, conservation and future studies in Dendrobium. We assessed the power of 11 candidate barcodes on the basis of 1,698 accessions of 184 Dendrobium species obtained primarily from mainland Asia. Our results indicated that five single barcodes, i.e., ITS, ITS2, matK, rbcL and trnH-psbA, can be easily amplified and sequenced with the currently established primers. Four barcodes, ITS, ITS2, ITS+matK, and ITS2+matK, have distinct barcoding gaps. ITS+matK was the optimal barcode based on all evaluation methods. Furthermore, the efficiency of ITS+matK was verified in four other large genera including Ficus, Lysimachia, Paphiopedilum, and Pedicularis in this study. Therefore, we tentatively recommend the combination of ITS+matK as a core DNA barcode for large flowering plant genera.

Evaluation of the DNA Barcodes in Dendrobium (Orchidaceae) from Mainland Asia

PubMed Central

Xu, Songzhi; Li, Dezhu; Li, Jianwu; Xiang, Xiaoguo; Jin, Weitao; Huang, Weichang; Jin, Xiaohua; Huang, Luqi

2015-01-01

DNA barcoding has been proposed to be one of the most promising tools for accurate and rapid identification of taxa. However, few publications have evaluated the efficiency of DNA barcoding for the large genera of flowering plants. Dendrobium, one of the largest genera of flowering plants, contains many species that are important in horticulture, medicine and biodiversity conservation. Besides, Dendrobium is a notoriously difficult group to identify. DNA barcoding was expected to be a supplementary means for species identification, conservation and future studies in Dendrobium. We assessed the power of 11 candidate barcodes on the basis of 1,698 accessions of 184 Dendrobium species obtained primarily from mainland Asia. Our results indicated that five single barcodes, i.e., ITS, ITS2, matK, rbcL and trnH-psbA, can be easily amplified and sequenced with the currently established primers. Four barcodes, ITS, ITS2, ITS+matK, and ITS2+matK, have distinct barcoding gaps. ITS+matK was the optimal barcode based on all evaluation methods. Furthermore, the efficiency of ITS+matK was verified in four other large genera including Ficus, Lysimachia, Paphiopedilum, and Pedicularis in this study. Therefore, we tentatively recommend the combination of ITS+matK as a core DNA barcode for large flowering plant genera. PMID:25602282

Species-specific identification from incomplete sampling: applying DNA barcodes to monitoring invasive solanum plants.

PubMed

Zhang, Wei; Fan, Xiaohong; Zhu, Shuifang; Zhao, Hong; Fu, Lianzhong

2013-01-01

Comprehensive sampling is crucial to DNA barcoding, but it is rarely performed because materials are usually unavailable. In practice, only a few rather than all species of a genus are required to be identified. Thus identification of a given species using a limited sample is of great importance in current application of DNA barcodes. Here, we selected 70 individuals representing 48 species from each major lineage of Solanum, one of the most species-rich genera of seed plants, to explore whether DNA barcodes can provide reliable specific-species discrimination in the context of incomplete sampling. Chloroplast genes ndhF and trnS-trnG and the nuclear gene waxy, the commonly used markers in Solanum phylogeny, were selected as the supplementary barcodes. The tree-building and modified barcode gap methods were employed to assess species resolution. The results showed that four Solanum species of quarantine concern could be successfully identified through the two-step barcoding sampling strategy. In addition, discrepancies between nuclear and cpDNA barcodes in some samples demonstrated the ability to discriminate hybrid species, and highlights the necessity of using barcode regions with different modes of inheritance. We conclude that efficient phylogenetic markers are good candidates as the supplementary barcodes in a given taxonomic group. Critically, we hypothesized that a specific-species could be identified from a phylogenetic framework using incomplete sampling-through this, DNA barcoding will greatly benefit the current fields of its application.

Expert Systems for Libraries at SCIL [Small Computers in Libraries]'88.

ERIC Educational Resources Information Center

Kochtanek, Thomas R.; And Others

1988-01-01

Six brief papers on expert systems for libraries cover (1) a knowledge-based approach to database design; (2) getting started in expert systems; (3) using public domain software to develop a business reference system; (4) a music cataloging inquiry system; (5) linguistic analysis of reference transactions; and (6) a model of a reference librarian.…

Improving the Virtual Reference Experience: How Closely Do Academic Libraries Adhere to RUSA Guidelines?

ERIC Educational Resources Information Center

Platt, Jessica; Benson, Pete

2010-01-01

The purpose of this study was to measure the degree to which academic libraries or library staff members throughout the United States adhere to the Guidelines for Virtual Reference Services provided by the Reference & User Services Association (RUSA). The results of the study were analyzed to identify specific areas where improvement is needed…

Resource Delivery and Teaching in Live Chat Reference: Comparing Two Libraries

ERIC Educational Resources Information Center

Dempsey, Paula R.

2017-01-01

This study investigates how reference staff at two libraries balance teaching with resource delivery in live chat reference. Analysis of 410 transcripts from one week shows that one library tends to deliver more resources from a wider range of database suggestions, to take more time in chat interactions, and to incorporate more teaching behavior…

Changes in Library Technology and Reference Desk Statistics: Is There a Relationship?

ERIC Educational Resources Information Center

Thomsett-Scott, Beth; Reese, Patricia E.

2006-01-01

The incorporation of technology into library processes has tremendously impacted staff and users alike. The University of North Texas (UNT) Libraries is no exception. Sixteen years of reference statistics are analyzed to examine the relationships between the implementation of CD-ROMs and web-based resources and the number of reference questions.…

Content Knowledge of the Internet Among Public Reference Librarians at the Cleveland Heights Public Library.

ERIC Educational Resources Information Center

Augustine, Matthew J.

The Internet is becoming an increasingly important medium of electronic information dissemination, and thus an increasingly important library reference tool. This study examines the Internet skills of a sample group of 15 public reference librarians in the Adult Services Department at the Cleveland Heights Public Library. The Internet skills that…

Does a global DNA barcoding gap exist in Annelida?

PubMed

Kvist, Sebastian

2016-05-01

Accurate identification of unknown specimens by means of DNA barcoding is contingent on the presence of a DNA barcoding gap, among other factors, as its absence may result in dubious specimen identifications - false negatives or positives. Whereas the utility of DNA barcoding would be greatly reduced in the absence of a distinct and sufficiently sized barcoding gap, the limits of intraspecific and interspecific distances are seldom thoroughly inspected across comprehensive sampling. The present study aims to illuminate this aspect of barcoding in a comprehensive manner for the animal phylum Annelida. All cytochrome c oxidase subunit I sequences (cox1 gene; the chosen region for zoological DNA barcoding) present in GenBank for Annelida, as well as for "Polychaeta", "Oligochaeta", and Hirudinea separately, were downloaded and curated for length, coverage and potential contaminations. The final datasets consisted of 9782 (Annelida), 5545 ("Polychaeta"), 3639 ("Oligochaeta"), and 598 (Hirudinea) cox1 sequences and these were either (i) used as is in an automated global barcoding gap detection analysis or (ii) further analyzed for genetic distances, separated into bins containing intraspecific and interspecific comparisons and plotted in a graph to visualize any potential global barcoding gap. Over 70 million pairwise genetic comparisons were made and results suggest that although there is a tendency towards separation, no distinct or sufficiently sized global barcoding gap exists in either of the datasets rendering future barcoding efforts at risk of erroneous specimen identifications (but local barcoding gaps may still exist allowing for the identification of specimens at lower taxonomic ranks). This seems to be especially true for earthworm taxa, which account for fully 35% of the total number of interspecific comparisons that show 0% divergence.

The chaperonin-60 universal target is a barcode for bacteria that enables de novo assembly of metagenomic sequence data.

PubMed

Links, Matthew G; Dumonceaux, Tim J; Hemmingsen, Sean M; Hill, Janet E

2012-01-01

Barcoding with molecular sequences is widely used to catalogue eukaryotic biodiversity. Studies investigating the community dynamics of microbes have relied heavily on gene-centric metagenomic profiling using two genes (16S rRNA and cpn60) to identify and track Bacteria. While there have been criteria formalized for barcoding of eukaryotes, these criteria have not been used to evaluate gene targets for other domains of life. Using the framework of the International Barcode of Life we evaluated DNA barcodes for Bacteria. Candidates from the 16S rRNA gene and the protein coding cpn60 gene were evaluated. Within complete bacterial genomes in the public domain representing 983 species from 21 phyla, the largest difference between median pairwise inter- and intra-specific distances ("barcode gap") was found from cpn60. Distribution of sequence diversity along the ∼555 bp cpn60 target region was remarkably uniform. The barcode gap of the cpn60 universal target facilitated the faithful de novo assembly of full-length operational taxonomic units from pyrosequencing data from a synthetic microbial community. Analysis supported the recognition of both 16S rRNA and cpn60 as DNA barcodes for Bacteria. The cpn60 universal target was found to have a much larger barcode gap than 16S rRNA suggesting cpn60 as a preferred barcode for Bacteria. A large barcode gap for cpn60 provided a robust target for species-level characterization of data. The assembly of consensus sequences for barcodes was shown to be a reliable method for the identification and tracking of novel microbes in metagenomic studies.

Combining and Comparing Coalescent, Distance and Character-Based Approaches for Barcoding Microalgaes: A Test with Chlorella-Like Species (Chlorophyta).

PubMed

Zou, Shanmei; Fei, Cong; Song, Jiameng; Bao, Yachao; He, Meilin; Wang, Changhai

2016-01-01

Several different barcoding methods of distinguishing species have been advanced, but which method is the best is still controversial. Chlorella is becoming particularly promising in the development of second-generation biofuels. However, the taxonomy of Chlorella-like organisms is easily confused. Here we report a comprehensive barcoding analysis of Chlorella-like species from Chlorella, Chloroidium, Dictyosphaerium and Actinastrum based on rbcL, ITS, tufA and 16S sequences to test the efficiency of traditional barcoding, GMYC, ABGD, PTP, P ID and character-based barcoding methods. First of all, the barcoding results gave new insights into the taxonomic assessment of Chlorella-like organisms studied, including the clear species discrimination and resolution of potentially cryptic species complexes in C. sorokiniana, D. ehrenbergianum and C. Vulgaris. The tufA proved to be the most efficient barcoding locus, which thus could be as potential "specific barcode" for Chlorella-like species. The 16S failed in discriminating most closely related species. The resolution of GMYC, PTP, P ID, ABGD and character-based barcoding methods were variable among rbcL, ITS and tufA genes. The best resolution for species differentiation appeared in tufA analysis where GMYC, PTP, ABGD and character-based approaches produced consistent groups while the PTP method over-split the taxa. The character analysis of rbcL, ITS and tufA sequences could clearly distinguish all taxonomic groups respectively, including the potentially cryptic lineages, with many character attributes. Thus, the character-based barcoding provides an attractive complement to coalescent and distance-based barcoding. Our study represents the test that proves the efficiency of multiple DNA barcoding in species discrimination of microalgaes.

Bilingual Virtual Reference: It's Better than Searching the Open Web

ERIC Educational Resources Information Center

Lupien, Pascal

2004-01-01

Online library services have been mostly available uniquely in English. To serve the diverse communities that uses library services, a growing number of libraries have been investigating the ins and outs of offering virtual reference in languages other than English. Developing this kind of service in languages spoken by various library user groups…

Benchmarking Reference Desk Service in Academic Health Science Libraries: A Preliminary Survey.

ERIC Educational Resources Information Center

Robbins, Kathryn; Daniels, Kathleen

2001-01-01

This preliminary study was designed to benchmark patron perceptions of reference desk services at academic health science libraries, using a standard questionnaire. Responses were compared to determine the library that provided the highest-quality service overall and along five service dimensions. All libraries were rated very favorably, but none…

Electronic Resource Expenditure and the Decline in Reference Transaction Statistics in Academic Libraries

ERIC Educational Resources Information Center

Dubnjakovic, Ana

2012-01-01

The current study investigates factors influencing increase in reference transactions in a typical week in academic libraries across the United States of America. Employing multiple regression analysis and general linear modeling, variables of interest from the "Academic Library Survey (ALS) 2006" survey (sample size 3960 academic libraries) were…

CD-ROM as a Library Equivalent.

ERIC Educational Resources Information Center

Reid, Harold T.

1992-01-01

Provides a brief overview of CD-ROM products that emulate a library's print collection. Highlights include reference works, including "Microsoft Bookshelf" and "Toolworks Reference Library"; the "New Grolier Electronic Encyclopedia"; periodicals and news services, including "Magazine Rack" and "Front…

DNA Barcoding of genus Hexacentrus in China reveals cryptic diversity within Hexacentrus japonicus (Orthoptera, Tettigoniidae).

PubMed

Guo, Hui-Fang; Guan, Bei; Shi, Fu-Ming; Zhou, Zhi-Jun

2016-01-01

DNA barcoding has been proved successful to provide resolution beyond the boundaries of morphological information. Hence, a study was undertaken to establish DNA barcodes for all morphologically determined Hexacentrus species in China collections. In total, 83 specimens of five Hexacentrus species were barcoded using standard mitochondrial cytochrome c oxidase subunit I (COI) gene. Except for Hexacentrus japonicus, barcode gaps were present in the remaining Hexacentrus species. Taxon ID tree generated seven BOLD's barcode index numbers (BINs), four of which were in agreement with the morphological species. For Hexacentrus japonicus, the maximum intraspecific divergence (4.43%) produced a minimal overlap (0.64%), and 19 specimens were divided into three different BINs. There may be cryptic species within the current Hexacentrus japonicus. This study adds to a growing body of DNA barcodes that have become available for katydids, and shows that a DNA barcoding approach enables the identification of known Hexacentrus species with a very high resolution.

DNA Barcoding of genus Hexacentrus in China reveals cryptic diversity within Hexacentrus japonicus (Orthoptera, Tettigoniidae)

PubMed Central

Guo, Hui-Fang; Guan, Bei; Shi, Fu-Ming; Zhou, Zhi-Jun

2016-01-01

Abstract DNA barcoding has been proved successful to provide resolution beyond the boundaries of morphological information. Hence, a study was undertaken to establish DNA barcodes for all morphologically determined Hexacentrus species in China collections. In total, 83 specimens of five Hexacentrus species were barcoded using standard mitochondrial cytochrome c oxidase subunit I (COI) gene. Except for Hexacentrus japonicus, barcode gaps were present in the remaining Hexacentrus species. Taxon ID tree generated seven BOLD’s barcode index numbers (BINs), four of which were in agreement with the morphological species. For Hexacentrus japonicus, the maximum intraspecific divergence (4.43%) produced a minimal overlap (0.64%), and 19 specimens were divided into three different BINs. There may be cryptic species within the current Hexacentrus japonicus. This study adds to a growing body of DNA barcodes that have become available for katydids, and shows that a DNA barcoding approach enables the identification of known Hexacentrus species with a very high resolution. PMID:27408576

Photocleavable DNA barcode-antibody conjugates allow sensitive and multiplexed protein analysis in single cells.

PubMed

Agasti, Sarit S; Liong, Monty; Peterson, Vanessa M; Lee, Hakho; Weissleder, Ralph

2012-11-14

DNA barcoding is an attractive technology, as it allows sensitive and multiplexed target analysis. However, DNA barcoding of cellular proteins remains challenging, primarily because barcode amplification and readout techniques are often incompatible with the cellular microenvironment. Here we describe the development and validation of a photocleavable DNA barcode-antibody conjugate method for rapid, quantitative, and multiplexed detection of proteins in single live cells. Following target binding, this method allows DNA barcodes to be photoreleased in solution, enabling easy isolation, amplification, and readout. As a proof of principle, we demonstrate sensitive and multiplexed detection of protein biomarkers in a variety of cancer cells.

Choosing and Using a Plant DNA Barcode

PubMed Central

Hollingsworth, Peter M.; Graham, Sean W.; Little, Damon P.

2011-01-01

The main aim of DNA barcoding is to establish a shared community resource of DNA sequences that can be used for organismal identification and taxonomic clarification. This approach was successfully pioneered in animals using a portion of the cytochrome oxidase 1 (CO1) mitochondrial gene. In plants, establishing a standardized DNA barcoding system has been more challenging. In this paper, we review the process of selecting and refining a plant barcode; evaluate the factors which influence the discriminatory power of the approach; describe some early applications of plant barcoding and summarise major emerging projects; and outline tool development that will be necessary for plant DNA barcoding to advance. PMID:21637336

Barcodes for genomes and applications

PubMed Central

Zhou, Fengfeng; Olman, Victor; Xu, Ying

2008-01-01

Background Each genome has a stable distribution of the combined frequency for each k-mer and its reverse complement measured in sequence fragments as short as 1000 bps across the whole genome, for 1

Half of the European fruit fly species barcoded (Diptera, Tephritidae); a feasibility test for molecular identification

PubMed Central

Smit, John; Reijnen, Bastian; Stokvis, Frank

2013-01-01

Abstract A feasibility test of molecular identification of European fruit flies (Diptera: Tephritidae) based on COI barcode sequences has been executed. A dataset containing 555 sequences of 135 ingroup species from three subfamilies and 42 genera and one single outgroup species has been analysed. 73.3% of all included species could be identified based on their COI barcode gene, based on similarity and distances. The low success rate is caused by singletons as well as some problematic groups: several species groups within the genus Terellia and especially the genus Urophora. With slightly more than 100 sequences – almost 20% of the total – this genus alone constitutes the larger part of the failure for molecular identification for this dataset. Deleting the singletons and Urophora results in a success-rate of 87.1% of all queries and 93.23% of the not discarded queries as correctly identified. Urophora is of special interest due to its economic importance as beneficial species for weed control, therefore it is desirable to have alternative markers for molecular identification. We demonstrate that the success of DNA barcoding for identification purposes strongly depends on the contents of the database used to BLAST against. Especially the necessity of including multiple specimens per species of geographically distinct populations and different ecologies for the understanding of the intra- versus interspecific variation is demonstrated. Furthermore thresholds and the distinction between true and false positives and negatives should not only be used to increase the reliability of the success of molecular identification but also to point out problematic groups, which should then be flagged in the reference database suggesting alternative methods for identification. PMID:24453563

Monitoring an alien invasion: DNA barcoding and the identification of lionfish and their prey on coral reefs of the Mexican Caribbean.

PubMed

Valdez-Moreno, Martha; Quintal-Lizama, Carolina; Gómez-Lozano, Ricardo; García-Rivas, María Del Carmen

2012-01-01

In the Mexican Caribbean, the exotic lionfish Pterois volitans has become a species of great concern because of their predatory habits and rapid expansion onto the Mesoamerican coral reef, the second largest continuous reef system in the world. This is the first report of DNA identification of stomach contents of lionfish using the barcode of life reference database (BOLD). We confirm with barcoding that only Pterois volitans is apparently present in the Mexican Caribbean. We analyzed the stomach contents of 157 specimens of P. volitans from various locations in the region. Based on DNA matches in the Barcode of Life Database (BOLD) and GenBank, we identified fishes from five orders, 14 families, 22 genera and 34 species in the stomach contents. The families with the most species represented were Gobiidae and Apogonidae. Some prey taxa are commercially important species. Seven species were new records for the Mexican Caribbean: Apogon mosavi, Coryphopterus venezuelae, C. thrix, C. tortugae, Lythrypnus minimus, Starksia langi and S. ocellata. DNA matches, as well as the presence of intact lionfish in the stomach contents, indicate some degree of cannibalism, a behavior confirmed in this species by the first time. We obtained 45 distinct crustacean prey sequences, from which only 20 taxa could be identified from the BOLD and GenBank databases. The matches were primarily to Decapoda but only a single taxon could be identified to the species level, Euphausia americana. This technique proved to be an efficient and useful method, especially since prey species could be identified from partially-digested remains. The primary limitation is the lack of comprehensive coverage of potential prey species in the region in the BOLD and GenBank databases, especially among invertebrates.

Monitoring an Alien Invasion: DNA Barcoding and the Identification of Lionfish and Their Prey on Coral Reefs of the Mexican Caribbean

PubMed Central

Valdez-Moreno, Martha; Quintal-Lizama, Carolina; Gómez-Lozano, Ricardo; García-Rivas, María del Carmen

2012-01-01

Background In the Mexican Caribbean, the exotic lionfish Pterois volitans has become a species of great concern because of their predatory habits and rapid expansion onto the Mesoamerican coral reef, the second largest continuous reef system in the world. This is the first report of DNA identification of stomach contents of lionfish using the barcode of life reference database (BOLD). Methodology/Principal Findings We confirm with barcoding that only Pterois volitans is apparently present in the Mexican Caribbean. We analyzed the stomach contents of 157 specimens of P. volitans from various locations in the region. Based on DNA matches in the Barcode of Life Database (BOLD) and GenBank, we identified fishes from five orders, 14 families, 22 genera and 34 species in the stomach contents. The families with the most species represented were Gobiidae and Apogonidae. Some prey taxa are commercially important species. Seven species were new records for the Mexican Caribbean: Apogon mosavi, Coryphopterus venezuelae, C. thrix, C. tortugae, Lythrypnus minimus, Starksia langi and S. ocellata. DNA matches, as well as the presence of intact lionfish in the stomach contents, indicate some degree of cannibalism, a behavior confirmed in this species by the first time. We obtained 45 distinct crustacean prey sequences, from which only 20 taxa could be identified from the BOLD and GenBank databases. The matches were primarily to Decapoda but only a single taxon could be identified to the species level, Euphausia americana. Conclusions/Significance This technique proved to be an efficient and useful method, especially since prey species could be identified from partially-digested remains. The primary limitation is the lack of comprehensive coverage of potential prey species in the region in the BOLD and GenBank databases, especially among invertebrates. PMID:22675470

Barcoded microchips for biomolecular assays.

PubMed

Zhang, Yi; Sun, Jiashu; Zou, Yu; Chen, Wenwen; Zhang, Wei; Xi, Jianzhong Jeff; Jiang, Xingyu

2015-01-20

Multiplexed assay of analytes is of great importance for clinical diagnostics and other analytical applications. Barcode-based bioassays with the ability to encode and decode may realize this goal in a straightforward and consistent manner. We present here a microfluidic barcoded chip containing several sets of microchannels with different widths, imitating the commonly used barcode. A single barcoded microchip can carry out tens of individual protein/nucleic acid assays (encode) and immediately yield all assay results by a portable barcode reader or a smartphone (decode). The applicability of a barcoded microchip is demonstrated by human immunodeficiency virus (HIV) immunoassays for simultaneous detection of three targets (anti-gp41 antibody, anti-gp120 antibody, and anti-gp36 antibody) from six human serum samples. We can also determine seven pathogen-specific oligonucleotides by a single chip containing both positive and negative controls.

Barcoding Sponges: An Overview Based on Comprehensive Sampling

PubMed Central

Vargas, Sergio; Schuster, Astrid; Sacher, Katharina; Büttner, Gabrielle; Schätzle, Simone; Läuchli, Benjamin; Hall, Kathryn; Hooper, John N. A.; Erpenbeck, Dirk; Wörheide, Gert

2012-01-01

Background Phylum Porifera includes ∼8,500 valid species distributed world-wide in aquatic ecosystems ranging from ephemeral fresh-water bodies to coastal environments and the deep-sea. The taxonomy and systematics of sponges is complicated, and morphological identification can be both time consuming and erroneous due to phenotypic convergence and secondary losses, etc. DNA barcoding can provide sponge biologists with a simple and rapid method for the identification of samples of unknown taxonomic membership. The Sponge Barcoding Project (www.spongebarcoding.org), the first initiative to barcode a non-bilaterian metazoan phylum, aims to provide a comprehensive DNA barcode database for Phylum Porifera. Methodology/Principal Findings ∼7,400 sponge specimens have been extracted, and amplification of the standard COI barcoding fragment has been attempted for approximately 3,300 museum samples with ∼25% mean amplification success. Based on this comprehensive sampling, we present the first report on the workflow and progress of the sponge barcoding project, and discuss some common pitfalls inherent to the barcoding of sponges. Conclusion A DNA-barcoding workflow capable of processing potentially large sponge collections has been developed and is routinely used for the Sponge Barcoding Project with success. Sponge specific problems such as the frequent co-amplification of non-target organisms have been detected and potential solutions are currently under development. The initial success of this innovative project have already demonstrated considerable refinement of sponge systematics, evaluating morphometric character importance, geographic phenotypic variability, and the utility of the standard barcoding fragment for Porifera (despite its conserved evolution within this basal metazoan phylum). PMID:22802937

The Role of Virtual Reference in Library Web Site Design: A Qualitative Source for Usage Data

ERIC Educational Resources Information Center

Powers, Amanda Clay; Shedd, Julie; Hill, Clay

2011-01-01

Gathering qualitative information about usage behavior of library Web sites is a time-consuming process requiring the active participation of patron communities. Libraries that collect virtual reference transcripts, however, hold valuable data regarding how the library Web site is used that could benefit Web designers. An analysis of virtual…

The Test of Reference.

ERIC Educational Resources Information Center

Childers, Thomas

1980-01-01

Reports the results of an unobtrusive study, from a user's viewpoint, of reference services available in the Suffolk Cooperative Library System. The study raises questions of policy centering around user expectations of library reference services. (RAA)

A Concealed Barcode Identification System Using Terahertz Time-domain Spectroscopy

NASA Astrophysics Data System (ADS)

Guan, Yu; Yamamoto, Manabu; Kitazawa, Toshiyuki; Tripathi, Saroj R.; Takeya, Kei; Kawase, Kodo

2015-03-01

We present a concealed terahertz barcode/chipless tag to achieve remote identification through an obstructing material using terahertz radiation. We show scanned terahertz reflection spectral images of barcodes concealed by a thick obstacle. A concealed and double- side printed terahertz barcode structure is proposed, and we demonstrate that our design has better performance in definition than a single-side printed barcode using terahertz time-domain spectroscopy. This technique combines the benefits of a chipless tag to read encoded information covered by an optically opaque material with low cost and a simple fabrication process. Simulations are also described, along with an explanation of the principle of the terahertz barcode identification system.

Rapidly evolving homing CRISPR barcodes

PubMed Central

Kalhor, Reza; Mali, Prashant; Church, George M.

2017-01-01

We present here an approach for engineering evolving DNA barcodes in living cells. The methodology entails using a homing guide RNA (hgRNA) scaffold that directs the Cas9-hgRNA complex to target the DNA locus of the hgRNA itself. We show that this homing CRISPR-Cas9 system acts as an expressed genetic barcode that diversifies its sequence and that the rate of diversification can be controlled in cultured cells. We further evaluate these barcodes in cell populations and show the barcode RNAs can be assayed as single molecules in situ . This integrated approach will have wide ranging applications, such as in deep lineage tracing, cellular barcoding, molecular recording, dissecting cancer biology, and connectome mapping. PMID:27918539

Generalisation and specialisation in hoverfly (Syrphidae) grassland pollen transport networks revealed by DNA metabarcoding.

PubMed

Lucas, Andrew; Bodger, Owen; Brosi, Berry J; Ford, Col R; Forman, Dan W; Greig, Carolyn; Hegarty, Matthew; Neyland, Penelope J; de Vere, Natasha

2018-04-16

Pollination by insects is a key ecosystem service and important to wider ecosystem function. Most species-level pollination networks studied have a generalised structure, with plants having several potential pollinators, and pollinators in turn visiting a number of different plant species. This is in apparent contrast to a plant's need for efficient conspecific pollen transfer. The aim of this study was to investigate the structure of pollen transport networks at three levels of biological hierarchy: community, species and individual. We did this using hoverflies in the genus Eristalis, a key group of non-Hymenopteran pollinators. We constructed pollen transport networks using DNA metabarcoding to identify pollen. We captured hoverflies in conservation grasslands in west Wales, UK, removed external pollen loads, sequenced the pollen DNA on the Illumina MiSeq platform using the standard plant barcode rbcL, and matched sequences using a pre-existing plant DNA barcode reference library. We found that Eristalis hoverflies transport pollen from 65 plant taxa, more than previously appreciated. Networks were generalised at the site and species level, suggesting some degree of functional redundancy, and were more generalised in late summer compared to early summer. In contrast, pollen transport at the individual level showed some degree of specialisation. Hoverflies defined as "single-plant visitors" varied from 40% of those captured in early summer to 24% in late summer. Individual hoverflies became more generalised in late summer, possibly in response to an increase in floral resources. Rubus fruticosus agg. and Succisa pratensis were key plant species for hoverflies at our sites Our results contribute to resolving the apparent paradox of how generalised pollinator networks can provide efficient pollination to plant species. Generalised hoverfly pollen transport networks may result from a varied range of short-term specialised feeding bouts by individual insects. The generalisation and functional redundancy of Eristalis pollen transport networks may increase the stability of the pollination service they deliver. © 2018 The Authors. Journal of Animal Ecology published by John Wiley & Sons Ltd on behalf of British Ecological Society.

[Integrated DNA barcoding database for identifying Chinese animal medicine].

PubMed

Shi, Lin-Chun; Yao, Hui; Xie, Li-Fang; Zhu, Ying-Jie; Song, Jing-Yuan; Zhang, Hui; Chen, Shi-Lin

2014-06-01

In order to construct an integrated DNA barcoding database for identifying Chinese animal medicine, the authors and their cooperators have completed a lot of researches for identifying Chinese animal medicines using DNA barcoding technology. Sequences from GenBank have been analyzed simultaneously. Three different methods, BLAST, barcoding gap and Tree building, have been used to confirm the reliabilities of barcode records in the database. The integrated DNA barcoding database for identifying Chinese animal medicine has been constructed using three different parts: specimen, sequence and literature information. This database contained about 800 animal medicines and the adulterants and closely related species. Unknown specimens can be identified by pasting their sequence record into the window on the ID page of species identification system for traditional Chinese medicine (www. tcmbarcode. cn). The integrated DNA barcoding database for identifying Chinese animal medicine is significantly important for animal species identification, rare and endangered species conservation and sustainable utilization of animal resources.

BOLDMirror: a global mirror system of DNA barcode data.

PubMed

Liu, D; Liu, L; Guo, G; Wang, W; Sun, Q; Parani, M; Ma, J

2013-11-01

DNA barcoding is a novel concept for taxonomic identification using short, specific genetic markers and has been applied to study a large number of eukaryotes. The huge amount of data output generated by DNA barcoding requires well-organized information systems. Besides the Barcode of Life Data system (BOLD) established in Canada, the mirror system is also important for the international barcode of life project (iBOL). For this purpose, we developed the BOLDMirror, a global mirror system of DNA barcode data. It is open-sourced and can run on the LAMP (Linux + Apache + MySQL + PHP) environment. BOLDMirror has data synchronization, data representation and statistics modules, and also provides spaces to store user operation history. BOLDMirror can be accessed at http://www.boldmirror.net and several countries have used it to setup their site of DNA barcoding. © 2012 John Wiley & Sons Ltd.

Insect barcode information system.

PubMed

Pratheepa, Maria; Jalali, Sushil Kumar; Arokiaraj, Robinson Silvester; Venkatesan, Thiruvengadam; Nagesh, Mandadi; Panda, Madhusmita; Pattar, Sharath

2014-01-01

Insect Barcode Information System called as Insect Barcode Informática (IBIn) is an online database resource developed by the National Bureau of Agriculturally Important Insects, Bangalore. This database provides acquisition, storage, analysis and publication of DNA barcode records of agriculturally important insects, for researchers specifically in India and other countries. It bridges a gap in bioinformatics by integrating molecular, morphological and distribution details of agriculturally important insects. IBIn was developed using PHP/My SQL by using relational database management concept. This database is based on the client- server architecture, where many clients can access data simultaneously. IBIn is freely available on-line and is user-friendly. IBIn allows the registered users to input new information, search and view information related to DNA barcode of agriculturally important insects.This paper provides a current status of insect barcode in India and brief introduction about the database IBIn. http://www.nabg-nbaii.res.in/barcode.

Barcoding Tetrahymena: discriminating species and identifying unknowns using the cytochrome c oxidase subunit I (cox-1) barcode.

PubMed

Kher, Chandni P; Doerder, F Paul; Cooper, Jason; Ikonomi, Pranvera; Achilles-Day, Undine; Küpper, Frithjof C; Lynn, Denis H

2011-01-01

DNA barcoding using the mitochondrial cytochromecoxidase subunit I (cox-1) gene has recently gained popularity as a tool for species identification of a variety of taxa. The primary objective of our research was to explore the efficacy of using cox-1 barcoding for species identification within the genusTetrahymena. We first increased intraspecific sampling forTetrahymena canadensis, Tetrahymena hegewischi, Tetrahymena pyriformis, Tetrahymena rostrata, Tetrahymena thermophila, and Tetrahymena tropicalis. Increased sampling efforts show that intraspecific sequence divergence is typically less than 1%, though it may be more in some species. The barcoding also showed that some strains might be misidentified or mislabeled. We also used cox-1 barcodes to provide species identifications for 51 unidentified environmental isolates, with a success rate of 98%. Thus, cox-1 barcoding is an invaluable tool for protistologists, especially when used in conjunction with morphological studies. 2010 Elsevier GmbH. All rights reserved.

Trends in reference usage statistics in an academic health sciences library.

PubMed

De Groote, Sandra L; Hitchcock, Kristin; McGowan, Richard

2007-01-01

To examine reference questions asked through traditional means at an academic health sciences library and place this data within the context of larger trends in reference services. Detailed data on the types of reference questions asked were collected during two one-month periods in 2003 and 2004. General statistics documenting broad categories of questions were compiled over a fifteen-year period. Administrative data show a steady increase in questions from 1990 to 1997/98 (23,848 to 48,037, followed by a decline through 2004/05 to 10,031. The distribution of reference questions asked over the years has changed-including a reduction in mediated searches 2,157 in 1990/91 to 18 in 2004/05, an increase in instruction 1,284 in 1993/94 to 1,897 in 2004/05 and an increase in digital reference interactions 0 in 1999/2000 to 581 in 2004/05. The most commonly asked questions at the current reference desk are about journal holdings 19%, book holdings 12%, and directional issues 12%. This study provides a unique snapshot of reference services in the contemporary library, where both online and offline services are commonplace. Changes in questions have impacted the way the library provides services, but traditional reference remains the core of information services in this health sciences library.

Systematic evaluation and optimization of modification reactions of oligonucleotides with amines and carboxylic acids for the synthesis of DNA-encoded chemical libraries.

PubMed

Franzini, Raphael M; Samain, Florent; Abd Elrahman, Maaly; Mikutis, Gediminas; Nauer, Angela; Zimmermann, Mauro; Scheuermann, Jörg; Hall, Jonathan; Neri, Dario

2014-08-20

DNA-encoded chemical libraries are collections of small molecules, attached to DNA fragments serving as identification barcodes, which can be screened against multiple protein targets, thus facilitating the drug discovery process. The preparation of large DNA-encoded chemical libraries crucially depends on the availability of robust synthetic methods, which enable the efficient conjugation to oligonucleotides of structurally diverse building blocks, sharing a common reactive group. Reactions of DNA derivatives with amines and/or carboxylic acids are particularly attractive for the synthesis of encoded libraries, in view of the very large number of building blocks that are commercially available. However, systematic studies on these reactions in the presence of DNA have not been reported so far. We first investigated conditions for the coupling of primary amines to oligonucleotides, using either a nucleophilic attack on chloroacetamide derivatives or a reductive amination on aldehyde-modified DNA. While both methods could be used for the production of secondary amines, the reductive amination approach was generally associated with higher yields and better purity. In a second endeavor, we optimized conditions for the coupling of a diverse set of 501 carboxylic acids to DNA derivatives, carrying primary and secondary amine functions. The coupling efficiency was generally higher for primary amines, compared to secondary amine substituents, but varied considerably depending on the structure of the acids and on the synthetic methods used. Optimal reaction conditions could be found for certain sets of compounds (with conversions >80%), but multiple reaction schemes are needed when assembling large libraries with highly diverse building blocks. The reactions and experimental conditions presented in this article should facilitate the synthesis of future DNA-encoded chemical libraries, while outlining the synthetic challenges that remain to be overcome.

78 FR 13006 - New Intelligent Mail Package Barcode Standards To Enhance Package Visibility; Opportunity for...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-02-26

...The Postal Service is exploring the advisability of requiring the use of Intelligent Mail[supreg] package barcodes (IMpb) or unique tracking Intelligent Mail barcodes (IMbTM) on all commercial parcels, and providing support to mailers to assure their ability to apply unique tracking barcodes to all commercial parcels.

DNA Barcoding of Marine Metazoa

NASA Astrophysics Data System (ADS)

Bucklin, Ann; Steinke, Dirk; Blanco-Bercial, Leocadio

2011-01-01

More than 230,000 known species representing 31 metazoan phyla populate the world's oceans. Perhaps another 1,000,000 or more species remain to be discovered. There is reason for concern that species extinctions may outpace discovery, especially in diverse and endangered marine habitats such as coral reefs. DNA barcodes (i.e., short DNA sequences for species recognition and discrimination) are useful tools to accelerate species-level analysis of marine biodiversity and to facilitate conservation efforts. This review focuses on the usual barcode region for metazoans: a ˜648 base-pair region of the mitochondrial cytochrome c oxidase subunit I (COI) gene. Barcodes have also been used for population genetic and phylogeographic analysis, identification of prey in gut contents, detection of invasive species, forensics, and seafood safety. More controversially, barcodes have been used to delimit species boundaries, reveal cryptic species, and discover new species. Emerging frontiers are the use of barcodes for rapid and increasingly automated biodiversity assessment by high-throughput sequencing, including environmental barcoding and the use of barcodes to detect species for which formal identification or scientific naming may never be possible.

Potential of DNA barcoding for detecting quarantine fungi.

PubMed

Gao, Ruifang; Zhang, Guiming

2013-11-01

The detection of live quarantine pathogenic fungi plays an important role in guaranteeing regional biological safety. DNA barcoding, an emerging species identification technology, holds promise for the reliable, quick, and accurate detection of quarantine fungi. International standards for phytosanitary guidelines are urgently needed. The varieties of quarantine fungi listed for seven countries/regions, the currently applied detection methods, and the status of DNA barcoding for detecting quarantine fungi are summarized in this study. Two approaches have been proposed to apply DNA barcoding to fungal quarantine procedures: (i) to verify the reliability of known internal transcribed spacer (ITS)/cytochrome c oxidase subunit I (COI) data for use as barcodes, and (ii) to determine other barcodes for species that cannot be identified by ITS/COI. As a unique, standardizable, and universal species identification tool, DNA barcoding offers great potential for integrating detection methods used in various countries/regions and establishing international detection standards based on accepted DNA barcodes. Through international collaboration, interstate disputes can be eased and many problems related to routine quarantine detection methods can be solved for global trade.

Designing robust watermark barcodes for multiplex long-read sequencing.

PubMed

Ezpeleta, Joaquín; Krsticevic, Flavia J; Bulacio, Pilar; Tapia, Elizabeth

2017-03-15

To attain acceptable sample misassignment rates, current approaches to multiplex single-molecule real-time sequencing require upstream quality improvement, which is obtained from multiple passes over the sequenced insert and significantly reduces the effective read length. In order to fully exploit the raw read length on multiplex applications, robust barcodes capable of dealing with the full single-pass error rates are needed. We present a method for designing sequencing barcodes that can withstand a large number of insertion, deletion and substitution errors and are suitable for use in multiplex single-molecule real-time sequencing. The manuscript focuses on the design of barcodes for full-length single-pass reads, impaired by challenging error rates in the order of 11%. The proposed barcodes can multiplex hundreds or thousands of samples while achieving sample misassignment probabilities as low as 10-7 under the above conditions, and are designed to be compatible with chemical constraints imposed by the sequencing process. Software tools for constructing watermark barcode sets and demultiplexing barcoded reads, together with example sets of barcodes and synthetic barcoded reads, are freely available at www.cifasis-conicet.gov.ar/ezpeleta/NS-watermark . ezpeleta@cifasis-conicet.gov.ar. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

An Algorithm Enabling Blind Users to Find and Read Barcodes

PubMed Central

Tekin, Ender; Coughlan, James M.

2010-01-01

Most camera-based systems for finding and reading barcodes are designed to be used by sighted users (e.g. the Red Laser iPhone app), and assume the user carefully centers the barcode in the image before the barcode is read. Blind individuals could benefit greatly from such systems to identify packaged goods (such as canned goods in a supermarket), but unfortunately in their current form these systems are completely inaccessible because of their reliance on visual feedback from the user. To remedy this problem, we propose a computer vision algorithm that processes several frames of video per second to detect barcodes from a distance of several inches; the algorithm issues directional information with audio feedback (e.g. “left,” “right”) and thereby guides a blind user holding a webcam or other portable camera to locate and home in on a barcode. Once the barcode is detected at sufficiently close range, a barcode reading algorithm previously developed by the authors scans and reads aloud the barcode and the corresponding product information. We demonstrate encouraging experimental results of our proposed system implemented on a desktop computer with a webcam held by a blindfolded user; ultimately the system will be ported to a camera phone for use by visually impaired users. PMID:20617114

DNA barcodes and citizen science provoke a diversity reappraisal for the "ring" butterflies of Peninsular Malaysia (Ypthima: Satyrinae: Nymphalidae: Lepidoptera).

PubMed

Jisming-See, Shi-Wei; Sing, Kong-Wah; Wilson, John-James

2016-10-01

The "rings" belonging to the genus Ypthima are amongst the most common butterflies in Peninsular Malaysia. However, the species can be difficult to tell apart, with keys relying on minor and often non-discrete ring characters found on the hindwing. Seven species have been reported from Peninsular Malaysia, but this is thought to be an underestimate of diversity. DNA barcodes of 165 individuals, and wing and genital morphology, were examined to reappraise species diversity of this genus in Peninsular Malaysia. DNA barcodes collected during citizen science projects-School Butterfly Project and Peninsular Malaysia Butterfly Count-recently conducted in Peninsular Malaysia were included. The new DNA barcodes formed six groups with different Barcode Index Numbers (BINs) representing four species reported in Peninsular Malaysia. When combined with public DNA barcodes from the Barcode Of Life Datasystems, several taxonomic issues arose. We consider the taxon Y. newboldi, formerly treated as a subspecies of Y. baldus, as a distinct species. DNA barcodes also supported an earlier suggestion that Y. nebulosa is a synonym under Y. horsfieldii humei. Two BINs of the genus Ypthima comprising DNA barcodes collected during citizen science projects did not correspond to any species previously reported in Peninsular Malaysia.

Reference Books in Special Media. Reference Circular No. 82-4.

ERIC Educational Resources Information Center

Library of Congress, Washington, DC. National Library Service for the Blind and Physically Handicapped.

Based on information contained in producers' catalogs and on responses to a survey conducted by the Reference Section of the Library of Congress National Library Service (NLS) for the Blind and Physically Handicapped, this publication lists reference materials produced in braille or in large type, and sound recordings of reference works available…

The Peer Reference Counseling Program at Odum Library. Training Manual.

ERIC Educational Resources Information Center

Lawrence, Tamiko Danielle; Thomas, Susan; Winston, Mark

The Peer Reference Counseling Program at Valdosta State University (Georgia) is a program designed to provide students with a unique opportunity to work at the Reference Desk at Odum Library. This program employs minority students and trains them to work at the Reference Desk answering basic reference questions and utilizing as well as…

FREQ-Seq: A Rapid, Cost-Effective, Sequencing-Based Method to Determine Allele Frequencies Directly from Mixed Populations

PubMed Central

Delaney, Nigel F.; Marx, Christopher J.

2012-01-01

Understanding evolutionary dynamics within microbial populations requires the ability to accurately follow allele frequencies through time. Here we present a rapid, cost-effective method (FREQ-Seq) that leverages Illumina next-generation sequencing for localized, quantitative allele frequency detection. Analogous to RNA-Seq, FREQ-Seq relies upon counts from the >105 reads generated per locus per time-point to determine allele frequencies. Loci of interest are directly amplified from a mixed population via two rounds of PCR using inexpensive, user-designed oligonucleotides and a bar-coded bridging primer system that can be regenerated in-house. The resulting bar-coded PCR products contain the adapters needed for Illumina sequencing, eliminating further library preparation. We demonstrate the utility of FREQ-Seq by determining the order and dynamics of beneficial alleles that arose as a microbial population, founded with an engineered strain of Methylobacterium, evolved to grow on methanol. Quantifying allele frequencies with minimal bias down to 1% abundance allowed effective analysis of SNPs, small in-dels and insertions of transposable elements. Our data reveal large-scale clonal interference during the early stages of adaptation and illustrate the utility of FREQ-Seq as a cost-effective tool for tracking allele frequencies in populations. PMID:23118913

CD133-targeted gene transfer into long-term repopulating hematopoietic stem cells.

PubMed

Brendel, Christian; Goebel, Benjamin; Daniela, Abriss; Brugman, Martijn; Kneissl, Sabrina; Schwäble, Joachim; Kaufmann, Kerstin B; Müller-Kuller, Uta; Kunkel, Hana; Chen-Wichmann, Linping; Abel, Tobias; Serve, Hubert; Bystrykh, Leonid; Buchholz, Christian J; Grez, Manuel

2015-01-01

Gene therapy for hematological disorders relies on the genetic modification of CD34(+) cells, a heterogeneous cell population containing about 0.01% long-term repopulating cells. Here, we show that the lentiviral vector CD133-LV, which uses a surface marker on human primitive hematopoietic stem cells (HSCs) as entry receptor, transfers genes preferentially into cells with high engraftment capability. Transduction of unstimulated CD34(+) cells with CD133-LV resulted in gene marking of cells with competitive proliferative advantage in vitro and in immunodeficient mice. The CD133-LV-transduced population contained significantly more cells with repopulating capacity than cells transduced with vesicular stomatitis virus (VSV)-LV, a lentiviral vector pseudotyped with the vesicular stomatitis virus G protein. Upon transfer of a barcode library, CD133-LV-transduced cells sustained gene marking in vivo for a prolonged period of time with a 6.7-fold higher recovery of barcodes compared to transduced control cells. Moreover, CD133-LV-transduced cells were capable of repopulating secondary recipients. Lastly, we show that this targeting strategy can be used for transfer of a therapeutic gene into CD34(+) cells obtained from patients suffering of X-linked chronic granulomatous disease. In conclusion, direct gene transfer into CD133(+) cells allows for sustained long-term engraftment of gene corrected cells.

The Real maccoyii: Identifying Tuna Sushi with DNA Barcodes – Contrasting Characteristic Attributes and Genetic Distances

PubMed Central

Lowenstein, Jacob H.; Amato, George; Kolokotronis, Sergios-Orestis

2009-01-01

Background The use of DNA barcodes for the identification of described species is one of the least controversial and most promising applications of barcoding. There is no consensus, however, as to what constitutes an appropriate identification standard and most barcoding efforts simply attempt to pair a query sequence with reference sequences and deem identification successful if it falls within the bounds of some pre-established cutoffs using genetic distance. Since the Renaissance, however, most biological classification schemes have relied on the use of diagnostic characters to identify and place species. Methodology/Principal Findings Here we developed a cytochrome c oxidase subunit I character-based key for the identification of all tuna species of the genus Thunnus, and compared its performance with distance-based measures for identification of 68 samples of tuna sushi purchased from 31 restaurants in Manhattan (New York City) and Denver, Colorado. Both the character-based key and GenBank BLAST successfully identified 100% of the tuna samples, while the Barcode of Life Database (BOLD) as well as genetic distance thresholds, and neighbor-joining phylogenetic tree building performed poorly in terms of species identification. A piece of tuna sushi has the potential to be an endangered species, a fraud, or a health hazard. All three of these cases were uncovered in this study. Nineteen restaurant establishments were unable to clarify or misrepresented what species they sold. Five out of nine samples sold as a variant of “white tuna” were not albacore (T. alalunga), but escolar (Lepidocybium flavorunneum), a gempylid species banned for sale in Italy and Japan due to health concerns. Nineteen samples were northern bluefin tuna (T. thynnus) or the critically endangered southern bluefin tuna (T. maccoyii), though nine restaurants that sold these species did not state these species on their menus. Conclusions/Significance The Convention on International Trade Endangered Species (CITES) requires that listed species must be identifiable in trade. This research fulfills this requirement for tuna, and supports the nomination of northern bluefin tuna for CITES listing in 2010. PMID:19924239

DNA barcoding gap: reliable species identification over morphological and geographical scales.

PubMed

Čandek, Klemen; Kuntner, Matjaž

2015-03-01

The philosophical basis and utility of DNA barcoding have been a subject of numerous debates. While most literature embraces it, some studies continue to question its use in dipterans, butterflies and marine gastropods. Here, we explore the utility of DNA barcoding in identifying spider species that vary in taxonomic affiliation, morphological diagnosibility and geographic distribution. Our first test searched for a 'barcoding gap' by comparing intra- and interspecific means, medians and overlap in more than 75,000 computed Kimura 2-parameter (K2P) genetic distances in three families. Our second test compared K2P distances of congeneric species with high vs. low morphological distinctness in 20 genera of 11 families. Our third test explored the effect of enlarging geographical sampling area at a continental scale on genetic variability in DNA barcodes within 20 species of nine families. Our results generally point towards a high utility of DNA barcodes in identifying spider species. However, the size of the barcoding gap strongly depends on taxonomic groups and practices. It is becoming critical to define the barcoding gap statistically more consistently and to document its variation over taxonomic scales. Our results support models of independent patterns of morphological and molecular evolution by showing that DNA barcodes are effective in species identification regardless of their morphological diagnosibility. We also show that DNA barcodes represent an effective tool for identifying spider species over geographic scales, yet their variation contains useful biogeographic information. © 2014 John Wiley & Sons Ltd.

A System for Anesthesia Drug Administration Using Barcode Technology: The Codonics Safe Label System and Smart Anesthesia Manager.

PubMed

Jelacic, Srdjan; Bowdle, Andrew; Nair, Bala G; Kusulos, Dolly; Bower, Lynnette; Togashi, Kei

2015-08-01

Many anesthetic drug errors result from vial or syringe swaps. Scanning the barcodes on vials before drug preparation, creating syringe labels that include barcodes, and scanning the syringe label barcodes before drug administration may help to prevent errors. In contrast, making syringe labels by hand that comply with the recommendations of regulatory agencies and standards-setting bodies is tedious and time consuming. A computerized system that uses vial barcodes and generates barcoded syringe labels could address both safety issues and labeling recommendations. We measured compliance of syringe labels in multiple operating rooms (ORs) with the recommendations of regulatory agencies and standards-setting bodies before and after the introduction of the Codonics Safe Label System (SLS). The Codonics SLS was then combined with Smart Anesthesia Manager software to create an anesthesia barcode drug administration system, which allowed us to measure the rate of scanning syringe label barcodes at the time of drug administration in 2 cardiothoracic ORs before and after introducing a coffee card incentive. Twelve attending cardiothoracic anesthesiologists and the OR satellite pharmacy participated. The use of the Codonics SLS drug labeling system resulted in >75% compliant syringe labels (95% confidence interval, 75%-98%). All syringe labels made using the Codonics SLS system were compliant. The average rate of scanning barcodes on syringe labels using Smart Anesthesia Manager was 25% (730 of 2976) over 13 weeks but increased to 58% (956 of 1645) over 8 weeks after introduction of a simple (coffee card) incentive (P < 0.001). An anesthesia barcode drug administration system resulted in a moderate rate of scanning syringe label barcodes at the time of drug administration. Further, adaptation of the system will be required to achieve a higher utilization rate.

Potential efficacy of mitochondrial genes for animal DNA barcoding: a case study using eutherian mammals.

PubMed

Luo, Arong; Zhang, Aibing; Ho, Simon Yw; Xu, Weijun; Zhang, Yanzhou; Shi, Weifeng; Cameron, Stephen L; Zhu, Chaodong

2011-01-28

A well-informed choice of genetic locus is central to the efficacy of DNA barcoding. Current DNA barcoding in animals involves the use of the 5' half of the mitochondrial cytochrome oxidase 1 gene (CO1) to diagnose and delimit species. However, there is no compelling a priori reason for the exclusive focus on this region, and it has been shown that it performs poorly for certain animal groups. To explore alternative mitochondrial barcoding regions, we compared the efficacy of the universal CO1 barcoding region with the other mitochondrial protein-coding genes in eutherian mammals. Four criteria were used for this comparison: the number of recovered species, sequence variability within and between species, resolution to taxonomic levels above that of species, and the degree of mutational saturation. Based on 1,179 mitochondrial genomes of eutherians, we found that the universal CO1 barcoding region is a good representative of mitochondrial genes as a whole because the high species-recovery rate (> 90%) was similar to that of other mitochondrial genes, and there were no significant differences in intra- or interspecific variability among genes. However, an overlap between intra- and interspecific variability was still problematic for all mitochondrial genes. Our results also demonstrated that any choice of mitochondrial gene for DNA barcoding failed to offer significant resolution at higher taxonomic levels. We suggest that the CO1 barcoding region, the universal DNA barcode, is preferred among the mitochondrial protein-coding genes as a molecular diagnostic at least for eutherian species identification. Nevertheless, DNA barcoding with this marker may still be problematic for certain eutherian taxa and our approach can be used to test potential barcoding loci for such groups.

Potential efficacy of mitochondrial genes for animal DNA barcoding: a case study using eutherian mammals

PubMed Central

2011-01-01

Background A well-informed choice of genetic locus is central to the efficacy of DNA barcoding. Current DNA barcoding in animals involves the use of the 5' half of the mitochondrial cytochrome oxidase 1 gene (CO1) to diagnose and delimit species. However, there is no compelling a priori reason for the exclusive focus on this region, and it has been shown that it performs poorly for certain animal groups. To explore alternative mitochondrial barcoding regions, we compared the efficacy of the universal CO1 barcoding region with the other mitochondrial protein-coding genes in eutherian mammals. Four criteria were used for this comparison: the number of recovered species, sequence variability within and between species, resolution to taxonomic levels above that of species, and the degree of mutational saturation. Results Based on 1,179 mitochondrial genomes of eutherians, we found that the universal CO1 barcoding region is a good representative of mitochondrial genes as a whole because the high species-recovery rate (> 90%) was similar to that of other mitochondrial genes, and there were no significant differences in intra- or interspecific variability among genes. However, an overlap between intra- and interspecific variability was still problematic for all mitochondrial genes. Our results also demonstrated that any choice of mitochondrial gene for DNA barcoding failed to offer significant resolution at higher taxonomic levels. Conclusions We suggest that the CO1 barcoding region, the universal DNA barcode, is preferred among the mitochondrial protein-coding genes as a molecular diagnostic at least for eutherian species identification. Nevertheless, DNA barcoding with this marker may still be problematic for certain eutherian taxa and our approach can be used to test potential barcoding loci for such groups. PMID:21276253

Assessment of Four Molecular Markers as Potential DNA Barcodes for Red Algae Kappaphycus Doty and Eucheuma J. Agardh (Solieriaceae, Rhodophyta)

PubMed Central

Tan, Ji; Lim, Phaik-Eem; Phang, Siew-Moi; Hong, Dang Diem; Sunarpi, H.; Hurtado, Anicia Q.

2012-01-01

DNA barcoding has been a major advancement in the field of taxonomy, seeing much effort put into the barcoding of wide taxa of organisms, macro and microalgae included. The mitochondrial-encoded cox1 and plastid-encoded rbcL has been proposed as potential DNA barcodes for rhodophytes, but are yet to be tested on the commercially important carrageenophytes Kappaphycus and Eucheuma. This study gauges the effectiveness of four markers, namely the mitochondrial cox1, cox2, cox2-3 spacer and the plastid rbcL in DNA barcoding on selected Kappaphycus and Eucheuma from Southeast Asia. Marker assessments were performed using established distance and tree-based identification criteria from earlier studies. Barcoding patterns on a larger scale were simulated by empirically testing on the commonly used cox2-3 spacer. The phylogeny of these rhodophytes was also briefly described. In this study, the cox2 marker which satisfies the prerequisites of DNA barcodes was found to exhibit moderately high interspecific divergences with no intraspecific variations, thus a promising marker for the DNA barcoding of Kappaphycus and Eucheuma. However, the already extensively used cox2-3 spacer was deemed to be in overall more appropriate as a DNA barcode for these two genera. On a wider scale, cox1 and rbcL were still better DNA barcodes across the rhodophyte taxa when practicality and cost-efficiency were taken into account. The phylogeny of Kappaphycus and Eucheuma were generally similar to those earlier reported. Still, the application of DNA barcoding has demonstrated our relatively poor taxonomic comprehension of these seaweeds, thus suggesting more in-depth efforts in taxonomic restructuring as well as establishment. PMID:23285223

Combining and Comparing Coalescent, Distance and Character-Based Approaches for Barcoding Microalgaes: A Test with Chlorella-Like Species (Chlorophyta)

PubMed Central

Zou, Shanmei; Fei, Cong; Song, Jiameng; Bao, Yachao; He, Meilin; Wang, Changhai

2016-01-01

Several different barcoding methods of distinguishing species have been advanced, but which method is the best is still controversial. Chlorella is becoming particularly promising in the development of second-generation biofuels. However, the taxonomy of Chlorella–like organisms is easily confused. Here we report a comprehensive barcoding analysis of Chlorella-like species from Chlorella, Chloroidium, Dictyosphaerium and Actinastrum based on rbcL, ITS, tufA and 16S sequences to test the efficiency of traditional barcoding, GMYC, ABGD, PTP, P ID and character-based barcoding methods. First of all, the barcoding results gave new insights into the taxonomic assessment of Chlorella-like organisms studied, including the clear species discrimination and resolution of potentially cryptic species complexes in C. sorokiniana, D. ehrenbergianum and C. Vulgaris. The tufA proved to be the most efficient barcoding locus, which thus could be as potential “specific barcode” for Chlorella-like species. The 16S failed in discriminating most closely related species. The resolution of GMYC, PTP, P ID, ABGD and character-based barcoding methods were variable among rbcL, ITS and tufA genes. The best resolution for species differentiation appeared in tufA analysis where GMYC, PTP, ABGD and character-based approaches produced consistent groups while the PTP method over-split the taxa. The character analysis of rbcL, ITS and tufA sequences could clearly distinguish all taxonomic groups respectively, including the potentially cryptic lineages, with many character attributes. Thus, the character-based barcoding provides an attractive complement to coalescent and distance-based barcoding. Our study represents the test that proves the efficiency of multiple DNA barcoding in species discrimination of microalgaes. PMID:27092945

1998 Idaho Public Library Statistics and Library Directory. A Compilation of Input and Output Measures and Other Statistics in Reference to Idaho's Public Libraries, Covering the Period October 1, 1997 to September 30, 1998.

ERIC Educational Resources Information Center

Nelson, Frank, Comp.

This report is a compilation of input and output measures and other statistics in reference to Idaho's public libraries, covering the period from October 1997 through September 1998. The introductory sections include notes on the statistics, definitions of performance measures, Idaho public library rankings for fiscal year 1996, and a state map…

Shift Colors

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Skip to main content Navigate Up This page location is: Navy Personnel Command Reference Library Command > Reference Library > Publications & News > Shift Colors Top Link Bar Navy Personnel Enlisted Support & Services Expand Support & Services Organization Expand Organization Reference

[Principles for molecular identification of traditional Chinese materia medica using DNA barcoding].

PubMed

Chen, Shi-Lin; Yao, Hui; Han, Jian-Ping; Xin, Tian-Yi; Pang, Xiao-Hui; Shi, Lin-Chun; Luo, Kun; Song, Jing-Yuan; Hou, Dian-Yun; Shi, Shang-Mei; Qian, Zhong-Zhi

2013-01-01

Since the research of molecular identification of Chinese Materia Medica (CMM) using DNA barcode is rapidly developing and popularizing, the principle of this method is approved to be listed in the Supplement of the Pharmacopoeia of the People's Republic of China. Based on the study on comprehensive samples, the DNA barcoding systems have been established to identify CMM, i.e. ITS2 as a core barcode and psbA-trnH as a complementary locus for identification of planta medica, and COI as a core barcode and ITS2 as a complementary locus for identification of animal medica. This article introduced the principle of molecular identification of CMM using DNA barcoding and its drafting instructions. Furthermore, its application perspective was discussed.

[Hydrophidae identification through analysis on Cyt b gene barcode].

PubMed

Liao, Li-xi; Zeng, Ke-wu; Tu, Peng-fei

2015-08-01

Hydrophidae, one of the precious traditional Chinese medicines, is generally drily preserved to prevent corruption, but it is hard to identify the species of Hydrophidae through the appearance because of the change due to the drying process. The identification through analysis on gene barcode, a new technique in species identification, can avoid the problem. The gene barcodes of the 6 species of Hydrophidae like Lapemis hardwickii were aquired through DNA extraction and gene sequencing. These barcodes were then in sequence alignment and test the identification efficency by BLAST. Our results revealed that the barcode sequences performed high identification efficiency, and had obvious difference between intra- and inter-species. These all indicated that Cyt b DNA barcoding can confirm the Hydrophidae identification.

Cost-effective ways of delivering enquiry services: a rapid review.

PubMed

Sutton, Anthea; Grant, Maria J

2011-12-01

In the recent times of recession and budget cuts, it is more important than ever for library and information services to deliver cost-effective services. This rapid review aims to examine the evidence for the most cost-effective ways of delivering enquiry services. A literature search was conducted on LISA (Library and Information Sciences Abstracts) and MEDLINE. Searches were limited to 2007 onwards. Eight studies met the inclusion criteria. The studies covered hospital and academic libraries in the USA and Canada. Services analysed were 'point-of-care' librarian consultations, staffing models for reference desks and virtual/digital reference services. Transferable lessons, relevant to health library and information services generally, can be drawn from this rapid review. These suggest that 'point-of-care' librarians for primary care practitioners are a cost-effective way of answering questions. Reference desks can be cost-effectively staffed by student employees or general reference staff, although librarian referral must be provided for more complex and subject-specific enquiries. However, it is not possible to draw any conclusions on virtual/digital reference services because of the limited literature available. Further case analysis studies measuring specific services, particularly enquiry services within a health library and information context, are required. © 2011 The authors. Health Information and Libraries Journal © 2011 Health Libraries Group.

Spectrum-to-Spectrum Searching Using a Proteome-wide Spectral Library*

PubMed Central

Yen, Chia-Yu; Houel, Stephane; Ahn, Natalie G.; Old, William M.

2011-01-01

The unambiguous assignment of tandem mass spectra (MS/MS) to peptide sequences remains a key unsolved problem in proteomics. Spectral library search strategies have emerged as a promising alternative for peptide identification, in which MS/MS spectra are directly compared against a reference library of confidently assigned spectra. Two problems relate to library size. First, reference spectral libraries are limited to rediscovery of previously identified peptides and are not applicable to new peptides, because of their incomplete coverage of the human proteome. Second, problems arise when searching a spectral library the size of the entire human proteome. We observed that traditional dot product scoring methods do not scale well with spectral library size, showing reduction in sensitivity when library size is increased. We show that this problem can be addressed by optimizing scoring metrics for spectrum-to-spectrum searches with large spectral libraries. MS/MS spectra for the 1.3 million predicted tryptic peptides in the human proteome are simulated using a kinetic fragmentation model (MassAnalyzer version2.1) to create a proteome-wide simulated spectral library. Searches of the simulated library increase MS/MS assignments by 24% compared with Mascot, when using probabilistic and rank based scoring methods. The proteome-wide coverage of the simulated library leads to 11% increase in unique peptide assignments, compared with parallel searches of a reference spectral library. Further improvement is attained when reference spectra and simulated spectra are combined into a hybrid spectral library, yielding 52% increased MS/MS assignments compared with Mascot searches. Our study demonstrates the advantages of using probabilistic and rank based scores to improve performance of spectrum-to-spectrum search strategies. PMID:21532008

Computer Aided Reference Services in the Academic Library: Experiences in Organizing and Operating an Online Reference Service.

ERIC Educational Resources Information Center

Hoover, Ryan E.

1979-01-01

Summarizes the development of the Computer-Aided Reference Services (CARS) division of the University of Utah Libraries' reference department. Development, organizational structure, site selection, equipment, management, staffing and training considerations, promotion and marketing, budget and pricing, record keeping, statistics, and evaluation…

The Librarian as Information Consultant: Transforming Reference for the Information Age

ERIC Educational Resources Information Center

Murphy, Sarah Anne

2011-01-01

Library users' evolving information needs and their choice of search methods have changed reference work profoundly. Today's reference librarian must work in a whole new way--not only service-focused and businesslike, but even entrepreneurial. Murphy innovatively rethinks the philosophy behind current library reference services in this…

Implementation and Use of the Reference Analytics Module of LibAnswers

ERIC Educational Resources Information Center

Flatley, Robert; Jensen, Robert Bruce

2012-01-01

Academic libraries have traditionally collected reference statistics using hash marks on paper. Although efficient and simple, this method is not an effective way to capture the complexity of reference transactions. Several electronic tools are now available to assist libraries with collecting often elusive reference data--among them homegrown…

Virtual Reference Services.

ERIC Educational Resources Information Center

Brewer, Sally

2003-01-01

As the need to access information increases, school librarians must create virtual libraries. Linked to reliable reference resources, the virtual library extends the physical collection and library hours and lets students learn to use Web-based resources in a protected learning environment. The growing number of virtual schools increases the need…

How to Survive in Industry. Cost Justifying Library Services

ERIC Educational Resources Information Center

Kramer, Joseph

1971-01-01

Two services provided by the Boeing Co. Aerospace Group Library-Literature searches and reference/publication identification activities-were evaluated by written and oral surveys of the library's users. The survey technique and cost savings reported by the two studies are discussed. (2 references) (Author/NH)

Comprehensive DNA barcoding of the herpetofauna of Germany.

PubMed

Hawlitschek, O; Morinière, J; Dunz, A; Franzen, M; Rödder, D; Glaw, F; Haszprunar, G

2016-01-01

We present the first comprehensive DNA barcoding study of German reptiles and amphibians representing likewise the first on the European herpetofauna. A total of 248 barcodes for all native species and subspecies in the country and a few additional taxa were obtained in the framework of the projects 'Barcoding Fauna Bavarica' (BFB) and 'German Barcode of Life' (GBOL). In contrast to many invertebrate groups, the success rate of the identification of mitochondrial lineages representing species via DNA barcode was almost 100% because no cases of Barcode Index Number (BIN) sharing were detected within German native reptiles and amphibians. However, as expected, a reliable identification of the hybridogenetic species complex in the frog genus Pelophylax was not possible. Deep conspecific lineages resulting in the identification of more than one BIN were found in Lissotriton vulgaris, Natrix natrix and the hybridogenetic Pelophylax complex. A high variety of lineages with different BINs was also found in the barcodes of wall lizards (Podarcis muralis), confirming the existence of many introduced lineages and the frequent occurrence of multiple introductions. Besides the reliable species identification of all life stages and even of tissue remains, our study highlights other potential applications of DNA barcoding concerning German amphibians and reptiles, such as the detection of allochthonous lineages, monitoring of gene flow and also noninvasive sampling via environmental DNA. DNA barcoding based on COI has now proven to be a reliable and efficient tool for studying most amphibians and reptiles as it is already for many other organism groups in zoology. © 2015 John Wiley & Sons Ltd.

Limitations of mitochondrial gene barcoding in Octocorallia.

PubMed

McFadden, Catherine S; Benayahu, Yehuda; Pante, Eric; Thoma, Jana N; Nevarez, P Andrew; France, Scott C

2011-01-01

The widespread assumption that COI and other mitochondrial genes will be ineffective DNA barcodes for anthozoan cnidarians has not been well tested for most anthozoans other than scleractinian corals. Here we examine the limitations of mitochondrial gene barcoding in the sub-class Octocorallia, a large, diverse, and ecologically important group of anthozoans. Pairwise genetic distance values (uncorrected p) were compared for three candidate barcoding regions: the Folmer region of COI; a fragment of the octocoral-specific mitochondrial protein-coding gene, msh1; and an extended barcode of msh1 plus COI with a short, adjacent intergenic region (igr1). Intraspecific variation was <0.5%, with most species exhibiting no variation in any of the three gene regions. Interspecific divergence was also low: 18.5% of congeneric morphospecies shared identical COI barcodes, and there was no discernible barcoding gap between intra- and interspecific p values. In a case study to assess regional octocoral biodiversity, COI and msh1 barcodes each identified 70% of morphospecies. In a second case study, a nucleotide character-based analysis correctly identified 70% of species in the temperate genus Alcyonium. Although interspecific genetic distances were 2× greater for msh1 than COI, each marker identified similar numbers of species in the two case studies, and the extended COI + igr1 + msh1 barcode more effectively discriminated sister taxa in Alcyonium. Although far from perfect for species identification, a COI + igr1 + msh1 barcode nonetheless represents a valuable addition to the depauperate set of characters available for octocoral taxonomy. © 2010 Blackwell Publishing Ltd.

The Mobile Reference Service: a case study of an onsite reference service program at the School of Public Health.

PubMed

Tao, Donghua; McCarthy, Patrick G; Krieger, Mary M; Webb, Annie B

2009-01-01

The School of Public Health at Saint Louis University is located at a greater distance from the library than other programs on the main medical center campus. Physical distance diminishes the ease of access to direct reference services for public health users. To bridge the gap, the library developed the Mobile Reference Service to deliver on-site information assistance with regular office hours each week. Between September 2006 and April 2007, a total of 57 in-depth reference transactions took place over 25 weeks, averaging 2 transactions per week in a 2-hour period. Overall reference transactions from public health users went up 28%, while liaison contacts with public health users doubled compared to the same period the year before. The Mobile Reference Service program has improved library support for research and scholarship, cultivated and strengthened liaison relationships, and enhanced marketing and delivery of library resources and services to the Saint Louis University School of Public Health.

The Mobile Reference Service: a case study of an onsite reference service program at the school of public health*

PubMed Central

Tao, Donghua; McCarthy, Patrick G.; Krieger, Mary M.; Webb, Annie B.

2009-01-01

The School of Public Health at Saint Louis University is located at a greater distance from the library than other programs on the main medical center campus. Physical distance diminishes the ease of access to direct reference services for public health users. To bridge the gap, the library developed the Mobile Reference Service to deliver onsite information assistance with regular office hours each week. Between September 2006 and April 2007, a total of 57 in-depth reference transactions took place over 25 weeks, averaging 2 transactions per week in a 2-hour period. Overall reference transactions from public health users went up 28%, while liaison contacts with public health users doubled compared to the same period the year before. The Mobile Reference Service program has improved library support for research and scholarship, cultivated and strengthened liaison relationships, and enhanced marketing and delivery of library resources and services to the Saint Louis University School of Public Health. PMID:19159004

Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species.

PubMed

Chen, Shilin; Yao, Hui; Han, Jianping; Liu, Chang; Song, Jingyuan; Shi, Linchun; Zhu, Yingjie; Ma, Xinye; Gao, Ting; Pang, Xiaohui; Luo, Kun; Li, Ying; Li, Xiwen; Jia, Xiaocheng; Lin, Yulin; Leon, Christine

2010-01-07

The plant working group of the Consortium for the Barcode of Life recommended the two-locus combination of rbcL+matK as the plant barcode, yet the combination was shown to successfully discriminate among 907 samples from 550 species at the species level with a probability of 72%. The group admits that the two-locus barcode is far from perfect due to the low identification rate, and the search is not over. Here, we compared seven candidate DNA barcodes (psbA-trnH, matK, rbcL, rpoC1, ycf5, ITS2, and ITS) from medicinal plant species. Our ranking criteria included PCR amplification efficiency, differential intra- and inter-specific divergences, and the DNA barcoding gap. Our data suggest that the second internal transcribed spacer (ITS2) of nuclear ribosomal DNA represents the most suitable region for DNA barcoding applications. Furthermore, we tested the discrimination ability of ITS2 in more than 6600 plant samples belonging to 4800 species from 753 distinct genera and found that the rate of successful identification with the ITS2 was 92.7% at the species level. The ITS2 region can be potentially used as a standard DNA barcode to identify medicinal plants and their closely related species. We also propose that ITS2 can serve as a novel universal barcode for the identification of a broader range of plant taxa.

Classification of Sharks in the Egyptian Mediterranean Waters Using Morphological and DNA Barcoding Approaches

PubMed Central

Moftah, Marie; Abdel Aziz, Sayeda H.; Elramah, Sara; Favereaux, Alexandre

2011-01-01

The identification of species constitutes the first basic step in phylogenetic studies, biodiversity monitoring and conservation. DNA barcoding, i.e. the sequencing of a short standardized region of DNA, has been proposed as a new tool for animal species identification. The present study provides an update on the composition of shark in the Egyptian Mediterranean waters off Alexandria, since the latest study to date was performed 30 years ago, DNA barcoding was used in addition to classical taxonomical methodologies. Thus, 51 specimen were DNA barcoded for a 667 bp region of the mitochondrial COI gene. Although DNA barcoding aims at developing species identification systems, some phylogenetic signals were apparent in the data. In the neighbor-joining tree, 8 major clusters were apparent, each of them containing individuals belonging to the same species, and most with 100% bootstrap value. This study is the first to our knowledge to use DNA barcoding of the mitochondrial COI gene in order to confirm the presence of species Squalus acanthias, Oxynotus centrina, Squatina squatina, Scyliorhinus canicula, Scyliorhinus stellaris, Mustelus mustelus, Mustelus punctulatus and Carcharhinus altimus in the Egyptian Mediterranean waters. Finally, our study is the starting point of a new barcoding database concerning shark composition in the Egyptian Mediterranean waters (Barcoding of Egyptian Mediterranean Sharks [BEMS], http://www.boldsystems.org/views/projectlist.php?&#Barcoding%20Fish%20%28FishBOL%29). PMID:22087242

Fixation, description and DNA barcode of a neotype for Botryllus schlosseri (Pallas, 1766) (Tunicata, Ascidiacea).

PubMed

Brunetti, Riccardo; Manni, Lucia; Mastrototaro, Francesco; Gissi, Carmela; Gasparini, Fabio

2017-11-22

Botryllus schlosseri is a widespread colonial ascidian commonly considered cosmopolitan and amply used as model for researches ranging from developmental biology to immunobiology. Recently, molecular data lead to hypothesize that the species named B. schlosseri may consist of more than a single taxon. Indeed, five highly divergent clades, named A-E, have been genetically identified and are referred as cryptic species. In this context, and lacking both a type and a detailed morphological description, we believe that it is necessary, as a taxonomic reference point, to designate a neotype and re-describe the species. Therefore, a sample from the Lagoon of Venice (Adriatic Sea, Italy) was deposited as neotype in the Natural History Museum of Venice (Italy), preserved both in formalin and in 90% ethanol. Here we provide a morphological description of the suggested neotype of B. schlosseri that takes into account several developmental stages (oozooid, zooid of first blastogenic generations, and mature zooid) and is carefully compared with the previous descriptions of samples coming from other European and non-European localities. Finally, we associate our morphological description to a "DNA barcode", consisting in a long fragment of the mitochondrial COI gene. Our description is associated to clade A, although at now we cannot guarantee that this association is univocal.

Cytochrome c oxidase I primers for corbiculate bees: DNA barcode and mini-barcode.

PubMed

Françoso, E; Arias, M C

2013-09-01

Bees (Apidae), of which there are more than 19 900 species, are extremely important for ecosystem services and economic purposes, so taxon identity is a major concern. The goal of this study was to optimize the DNA barcode technique based on the Cytochrome c oxidase (COI) mitochondrial gene region. This approach has previously been shown to be useful in resolving taxonomic inconsistencies and for species identification when morphological data are poor. Specifically, we designed and tested new primers and standardized PCR conditions to amplify the barcode region for bees, focusing on the corbiculate Apids. In addition, primers were designed to amplify small COI amplicons and tested with pinned specimens. Short barcode sequences were easily obtained for some Bombus century-old museum specimens and shown to be useful as mini-barcodes. The new primers and PCR conditions established in this study proved to be successful for the amplification of the barcode region for all species tested, regardless of the conditions of tissue preservation. We saw no evidence of Wolbachia or numts amplification by these primers, and so we suggest that these new primers are of broad value for corbiculate bee identification through DNA barcode. © 2013 John Wiley & Sons Ltd.

Automating quantum dot barcode assays using microfluidics and magnetism for the development of a point-of-care device.

PubMed

Gao, Yali; Lam, Albert W Y; Chan, Warren C W

2013-04-24

The impact of detecting multiple infectious diseases simultaneously at point-of-care with good sensitivity, specificity, and reproducibility would be enormous for containing the spread of diseases in both resource-limited and rich countries. Many barcoding technologies have been introduced for addressing this need as barcodes can be applied to detecting thousands of genetic and protein biomarkers simultaneously. However, the assay process is not automated and is tedious and requires skilled technicians. Barcoding technology is currently limited to use in resource-rich settings. Here we used magnetism and microfluidics technology to automate the multiple steps in a quantum dot barcode assay. The quantum dot-barcoded microbeads are sequentially (a) introduced into the chip, (b) magnetically moved to a stream containing target molecules, (c) moved back to the original stream containing secondary probes, (d) washed, and (e) finally aligned for detection. The assay requires 20 min, has a limit of detection of 1.2 nM, and can detect genetic targets for HIV, hepatitis B, and syphilis. This study provides a simple strategy to automate the entire barcode assay process and moves barcoding technologies one step closer to point-of-care applications.

Taxonomy, distribution and ecology of the order Phyllodocida (Annelida, Polychaeta) in deep-sea habitats around the Iberian margin

NASA Astrophysics Data System (ADS)

Ravara, Ascensão; Ramos, Diana; Teixeira, Marcos A. L.; Costa, Filipe O.; Cunha, Marina R.

2017-03-01

The polychaetes of the order Phyllodocida (excluding Nereidiformia and Phyllodociformia incertae sedis) collected from deep-sea habitats of the Iberian margin (Bay of Biscay, Horseshoe continental rise, Gulf of Cadiz and Alboran Sea), and Atlantic seamounts (Gorringe Bank, Atlantis and Nameless) are reported herein. Thirty-six species belonging to seven families - Acoetidae, Pholoidae, Polynoidae, Sigalionidae, Glyceridae, Goniadidae and Phyllodocidae, were identified. Amended descriptions and/or new illustrations are given for the species Allmaniella setubalensis, Anotochaetonoe michelbhaudi, Lepidasthenia brunnea and Polynoe sp. Relevant taxonomical notes are provided for other seventeen species. Allmaniella setubalensis, Anotochaetonoe michelbhaudi, Harmothoe evei, Eumida longicirrata and Glycera noelae, previously known only from their type localities were found in different deep-water places of the studied areas and constitute new records for the Iberian margin. The geographic distributions and the bathymetric range of thirteen and fifteen species, respectively, are extended. The morphology-based biodiversity inventory was complemented with DNA sequences of the mitochondrial barcode region (COI barcodes) providing a molecular tag for future reference. Twenty new sequences were obtained for nine species in the families Acoetidae, Glyceridae and Polynoidae and for three lineages within the Phylodoce madeirensis complex (Phyllodocidae). A brief analysis of the newly obtained sequences and publicly available COI barcode data for the genera herein reported, highlighted several cases of unclear taxonomic assignments, which need further study.

Using DNA Barcodes to Identify Road-Killed Animals in Two Atlantic Forest Nature Reserves, Brazil

PubMed Central

Klippel, Angélica H.; Oliveira, Pablo V.; Britto, Karollini B.; Freire, Bárbara F.; Moreno, Marcel R.; dos Santos, Alexandre R.; Banhos, Aureo; Paneto, Greiciane G.

2015-01-01

Road mortality is the leading source of biodiversity loss in the world, especially due to fragmentation of natural habitats and loss of wildlife. The survey of the main species victims of roadkill is of fundamental importance for the better understanding of the problem, being necessary, for this, the correct species identification. The aim of this study was to verify if DNA barcodes can be applied to identify road-killed samples that often cannot be determined morphologically. For this purpose, 222 vertebrate samples were collected in a stretch of the BR-101 highway that crosses two Discovery Coast Atlantic Forest Natural Reserves, the Sooretama Biological Reserve and the Vale Natural Reserve, in Espírito Santo, Brazil. The mitochondrial COI gene was amplified, sequenced and confronted with the BOLD database. It was possible to identify 62.16% of samples, totaling 62 different species, including Pyrrhura cruentata, Chaetomys subspinosus, Puma yagouaroundi and Leopardus wiedii considered Vulnerable in the National Official List of Species of Endangered Wildlife. The most commonly identified animals were a bat (Molossus molossus), an opossum (Didelphis aurita) and a frog (Trachycephalus mesophaeus) species. Only one reptile was identified using the technique, probably due to lack of reference sequences in BOLD. These data may contribute to a better understanding of the impact of roads on species biodiversity loss and to introduce the DNA barcode technique to road ecology scenarios. PMID:26244644

The Barcode of Life Data Portal: Bridging the Biodiversity Informatics Divide for DNA Barcoding

PubMed Central

Sarkar, Indra Neil; Trizna, Michael

2011-01-01

With the volume of molecular sequence data that is systematically being generated globally, there is a need for centralized resources for data exploration and analytics. DNA Barcode initiatives are on track to generate a compendium of molecular sequence–based signatures for identifying animals and plants. To date, the range of available data exploration and analytic tools to explore these data have only been available in a boutique form—often representing a frustrating hurdle for many researchers that may not necessarily have resources to install or implement algorithms described by the analytic community. The Barcode of Life Data Portal (BDP) is a first step towards integrating the latest biodiversity informatics innovations with molecular sequence data from DNA barcoding. Through establishment of community driven standards, based on discussion with the Data Analysis Working Group (DAWG) of the Consortium for the Barcode of Life (CBOL), the BDP provides an infrastructure for incorporation of existing and next-generation DNA barcode analytic applications in an open forum. PMID:21818249

DNA barcoding Indian freshwater fishes.

PubMed

Lakra, Wazir Singh; Singh, M; Goswami, Mukunda; Gopalakrishnan, A; Lal, K K; Mohindra, V; Sarkar, U K; Punia, P P; Singh, K V; Bhatt, J P; Ayyappan, S

2016-11-01

DNA barcoding is a promising technique for species identification using a short mitochondrial DNA sequence of cytochrome c oxidase I (COI) gene. In the present study, DNA barcodes were generated from 72 species of freshwater fish covering the Orders Cypriniformes, Siluriformes, Perciformes, Synbranchiformes, and Osteoglossiformes representing 50 genera and 19 families. All the samples were collected from diverse sites except the species endemic to a particular location. Species were represented by multiple specimens in the great majority of the barcoded species. A total of 284 COI sequences were generated. After amplification and sequencing of 700 base pair fragment of COI, primers were trimmed which invariably generated a 655 base pair barcode sequence. The average Kimura two-parameter (K2P) distances within-species, genera, families, and orders were 0.40%, 9.60%, 13.10%, and 17.16%, respectively. DNA barcode discriminated congeneric species without any confusion. The study strongly validated the efficiency of COI as an ideal marker for DNA barcoding of Indian freshwater fishes.

Using barcoded Zika virus to assess virus population structure in vitro and in Aedes aegypti mosquitoes.

PubMed

Weger-Lucarelli, James; Garcia, Selene M; Rückert, Claudia; Byas, Alex; O'Connor, Shelby L; Aliota, Matthew T; Friedrich, Thomas C; O'Connor, David H; Ebel, Gregory D

2018-06-20

Arboviruses such as Zika virus (ZIKV, Flaviviridae; Flavivirus) must replicate in both mammalian and insect hosts possessing strong immune defenses. Accordingly, transmission between and replication within hosts involves genetic bottlenecks, during which viral population size and genetic diversity may be significantly reduced. To help quantify these bottlenecks and their effects, we constructed 4 "barcoded" ZIKV populations that theoretically contain thousands of barcodes each. After identifying the most diverse barcoded virus, we passaged this virus 3 times in 2 mammalian and mosquito cell lines and characterized the population using deep sequencing of the barcoded region of the genome. C6/36 maintain higher barcode diversity, even after 3 passages, than Vero. Additionally, field-caught mosquitoes exposed to the virus to assess bottlenecks in a natural host. A progressive reduction in barcode diversity occurred throughout systemic infection of these mosquitoes. Differences in bottlenecks during systemic spread were observed between different populations of Aedes aegypti. Copyright © 2018. Published by Elsevier Inc.

Content Analysis of Virtual Reference Data: Reshaping Library Website Design.

PubMed

Fan, Suhua Caroline; Welch, Jennifer M

2016-01-01

An academic health sciences library wanted to redesign its website to provide better access to health information in the community. Virtual reference data were used to provide information about user searching behavior. This study analyzed three years (2012-2014) of virtual reference data, including e-mail questions, text messaging, and live chat transcripts, to evaluate the library website for redesigning, especially in areas such as the home page, patrons' terminology, and issues prompting patrons to ask for help. A coding system based on information links in the current library website was created to analyze the data.

DNA Barcoding of Neotropical Sand Flies (Diptera, Psychodidae, Phlebotominae): Species Identification and Discovery within Brazil

PubMed Central

Pinto, Israel de Souza; Chagas, Bruna Dias das; Rodrigues, Andressa Alencastre Fuzari; Ferreira, Adelson Luiz; Rezende, Helder Ricas; Bruno, Rafaela Vieira; Falqueto, Aloisio; Andrade-Filho, José Dilermando; Galati, Eunice Aparecida Bianchi; Shimabukuro, Paloma Helena Fernandes; Brazil, Reginaldo Peçanha

2015-01-01

DNA barcoding has been an effective tool for species identification in several animal groups. Here, we used DNA barcoding to discriminate between 47 morphologically distinct species of Brazilian sand flies. DNA barcodes correctly identified approximately 90% of the sampled taxa (42 morphologically distinct species) using clustering based on neighbor-joining distance, of which four species showed comparatively higher maximum values of divergence (range 4.23–19.04%), indicating cryptic diversity. The DNA barcodes also corroborated the resurrection of two species within the shannoni complex and provided an efficient tool to differentiate between morphologically indistinguishable females of closely related species. Taken together, our results validate the effectiveness of DNA barcoding for species identification and the discovery of cryptic diversity in sand flies from Brazil. PMID:26506007

DNA Barcoding of Neotropical Sand Flies (Diptera, Psychodidae, Phlebotominae): Species Identification and Discovery within Brazil.

PubMed

Pinto, Israel de Souza; Chagas, Bruna Dias das; Rodrigues, Andressa Alencastre Fuzari; Ferreira, Adelson Luiz; Rezende, Helder Ricas; Bruno, Rafaela Vieira; Falqueto, Aloisio; Andrade-Filho, José Dilermando; Galati, Eunice Aparecida Bianchi; Shimabukuro, Paloma Helena Fernandes; Brazil, Reginaldo Peçanha; Peixoto, Alexandre Afranio

2015-01-01

DNA barcoding has been an effective tool for species identification in several animal groups. Here, we used DNA barcoding to discriminate between 47 morphologically distinct species of Brazilian sand flies. DNA barcodes correctly identified approximately 90% of the sampled taxa (42 morphologically distinct species) using clustering based on neighbor-joining distance, of which four species showed comparatively higher maximum values of divergence (range 4.23-19.04%), indicating cryptic diversity. The DNA barcodes also corroborated the resurrection of two species within the shannoni complex and provided an efficient tool to differentiate between morphologically indistinguishable females of closely related species. Taken together, our results validate the effectiveness of DNA barcoding for species identification and the discovery of cryptic diversity in sand flies from Brazil.

Meeting Reference Responsibilities through Library Web Sites.

ERIC Educational Resources Information Center

Adams, Michael

2001-01-01

Discusses library Web sites and explains some of the benefits when libraries make their sites into reference portals, linking them to other useful Web sites. Topics include print versus Web information sources; limitations of search engines; what Web sites to include, including criteria for inclusions; and organizing the sites. (LRW)

Libraries & Study Facilities. A Selected Bibliography.

ERIC Educational Resources Information Center

Wisconsin Univ., Madison. ERIC Clearinghouse on Educational Facilities.

This bibliography contains a selected reference list of publications of interest to architects, library planners, and librarians contemplating the planning, programing, and/or design of library facilities. Each reference is followed by a listing of ERIC descriptors that describe its contents. The items are listed in six sections: (1) library…

Virtual Reference Service in Academic Libraries in West Africa

ERIC Educational Resources Information Center

Sekyere, Kwabena

2011-01-01

As technology continues to advance, libraries in Europe and America continue to improve upon their virtual reference services by employing new Web technologies and applying them to existing services. West African academic libraries have begun providing resources electronically to their users but still typically lag behind in the services they…

Young Adult Reference Services in the Public Library.

ERIC Educational Resources Information Center

Boylan, Patricia

1984-01-01

Methods suggested for use by public libraries to stay on top of school assignments include a large, loose-leaf type binder entitled "School Assignments" to be kept at reference desk; assignment-related book lists; school assignment forms; and teacher notification forms to alert them if the library cannot fulfill their information…

Closed-Tube Barcoding.

PubMed

Sirianni, Nicky M; Yuan, Huijun; Rice, John E; Kaufman, Ronit S; Deng, John; Fulton, Chandler; Wangh, Lawrence J

2016-11-01

Here, we present a new approach for increasing the rate and lowering the cost of identifying, cataloging, and monitoring global biodiversity. These advances, which we call Closed-Tube Barcoding, are one application of a suite of proven PCR-based technologies invented in our laboratory. Closed-Tube Barcoding builds on and aims to enhance the profoundly important efforts of the International Barcode of Life initiative. Closed-Tube Barcoding promises to be particularly useful when large numbers of small or rare specimens need to be screened and characterized at an affordable price. This approach is also well suited for automation and for use in portable devices.

Moving Reference to the Web.

ERIC Educational Resources Information Center

McGlamery, Susan; Coffman, Steve

2000-01-01

Explores the possibility of using Web contact center software to offer reference assistance to remote users. Discusses a project by the Metropolitan Cooperative Library System/Santiago Library System consortium to test contact center software and to develop a virtual reference network. (Author/LRW)

Moving beyond Assumptions: The Use of Virtual Reference Data in an Academic Library

ERIC Educational Resources Information Center

Nolen, David S.; Powers, Amanda Clay; Zhang, Li; Xu, Yue; Cannady, Rachel E.; Li, Judy

2012-01-01

The Mississippi State University Libraries' Virtual Reference Service collected statistics about virtual reference usage. Analysis of the data collected by an entry survey from chat and e-mail transactions provided librarians with concrete information about what patron groups were the highest and lowest users of virtual reference services. These…

Recommended Reference Books for Small and Medium-Sized Libraries and Media Centers, 1999.

ERIC Educational Resources Information Center

Wynar, Bohdan S., Ed.

Designed to assist smaller libraries in selecting suitable reference materials for their collections, this annual review source identifies and describes 538 of the most useful and affordable reference sources available. The reviews cover reference titles published in 1998. Detailed annotations describe the nature, scope, and usability of each…

Supervised DNA Barcodes species classification: analysis, comparisons and results

PubMed Central

2014-01-01

Background Specific fragments, coming from short portions of DNA (e.g., mitochondrial, nuclear, and plastid sequences), have been defined as DNA Barcode and can be used as markers for organisms of the main life kingdoms. Species classification with DNA Barcode sequences has been proven effective on different organisms. Indeed, specific gene regions have been identified as Barcode: COI in animals, rbcL and matK in plants, and ITS in fungi. The classification problem assigns an unknown specimen to a known species by analyzing its Barcode. This task has to be supported with reliable methods and algorithms. Methods In this work the efficacy of supervised machine learning methods to classify species with DNA Barcode sequences is shown. The Weka software suite, which includes a collection of supervised classification methods, is adopted to address the task of DNA Barcode analysis. Classifier families are tested on synthetic and empirical datasets belonging to the animal, fungus, and plant kingdoms. In particular, the function-based method Support Vector Machines (SVM), the rule-based RIPPER, the decision tree C4.5, and the Naïve Bayes method are considered. Additionally, the classification results are compared with respect to ad-hoc and well-established DNA Barcode classification methods. Results A software that converts the DNA Barcode FASTA sequences to the Weka format is released, to adapt different input formats and to allow the execution of the classification procedure. The analysis of results on synthetic and real datasets shows that SVM and Naïve Bayes outperform on average the other considered classifiers, although they do not provide a human interpretable classification model. Rule-based methods have slightly inferior classification performances, but deliver the species specific positions and nucleotide assignments. On synthetic data the supervised machine learning methods obtain superior classification performances with respect to the traditional DNA Barcode classification methods. On empirical data their classification performances are at a comparable level to the other methods. Conclusions The classification analysis shows that supervised machine learning methods are promising candidates for handling with success the DNA Barcoding species classification problem, obtaining excellent performances. To conclude, a powerful tool to perform species identification is now available to the DNA Barcoding community. PMID:24721333

Barcode Technology Acceptance and Utilization in Health Information Management Department at Academic Hospitals According to Technology Acceptance Model

PubMed Central

Ehteshami, Asghar

2017-01-01

Nowdays, due to the increasing importance of quality care, organizations focuse on the improving provision, management and distribution of health. On one hand, incremental costs of the new technologies and on the other hand, increased knowledge of health care recipients and their expectations for high quality services have doubled the need to make changes in order to respond to resource constraints (financial, human, material). For this purpose, several technologies, such as barcode, have been used in hospitals to improve services and staff productivity; but various factors effect on the adoption of new technologies and despite good implementation of a technology and its benefits, sometimes personnel don’t accept and don’t use it. Methods: This is an applied descriptive cross-sectional study in which all the barcode users in health information management department of the three academic hospitals (Feiz, Al-Zahra, Ayatollah Kashani) affiliated to Isfahan University of Medical Sciences were surveyed by the barcode technology acceptance questionnaire, in six areas as following: barcode ease of learning, capabilities, perception of its usefulness and its ease of use, users attitudes towards its using, and users intention. Results: The finding showed that barcode technology total acceptance was relatively desirable (%76.9); the most compliance with TAM model was related to the user perceptions about the ease of use of barcode technology and the least compliance was related to the ease of learning barcode technology (respectively %83.7 and %71.5). Conclusion: Ease of learning and barcode capability effect of usefulness and perceived ease of barcode technology. Users perceptions effect their attitudes toward greater use of technology and their attitudes have an effect on their intention to use the technology and finally, their intention makes actual use of the technology (acceptance). Therefore, considering the six elements related to technology implementation can be important in the barcode acceptance; because their chained relationship is clearly visible. PMID:28484289

Barcode Technology Acceptance and Utilization in Health Information Management Department at Academic Hospitals According to Technology Acceptance Model.

PubMed

Ehteshami, Asghar

2017-03-01

Nowdays, due to the increasing importance of quality care, organizations focuse on the improving provision, management and distribution of health. On one hand, incremental costs of the new technologies and on the other hand, increased knowledge of health care recipients and their expectations for high quality services have doubled the need to make changes in order to respond to resource constraints (financial, human, material). For this purpose, several technologies, such as barcode, have been used in hospitals to improve services and staff productivity; but various factors effect on the adoption of new technologies and despite good implementation of a technology and its benefits, sometimes personnel don't accept and don't use it. This is an applied descriptive cross-sectional study in which all the barcode users in health information management department of the three academic hospitals (Feiz, Al-Zahra, Ayatollah Kashani) affiliated to Isfahan University of Medical Sciences were surveyed by the barcode technology acceptance questionnaire, in six areas as following: barcode ease of learning, capabilities, perception of its usefulness and its ease of use, users attitudes towards its using, and users intention. The finding showed that barcode technology total acceptance was relatively desirable (%76.9); the most compliance with TAM model was related to the user perceptions about the ease of use of barcode technology and the least compliance was related to the ease of learning barcode technology (respectively %83.7 and %71.5). Ease of learning and barcode capability effect of usefulness and perceived ease of barcode technology. Users perceptions effect their attitudes toward greater use of technology and their attitudes have an effect on their intention to use the technology and finally, their intention makes actual use of the technology (acceptance). Therefore, considering the six elements related to technology implementation can be important in the barcode acceptance; because their chained relationship is clearly visible.

Evaluation and Adaptation of a Laboratory-Based cDNA Library Preparation Protocol for Retrospective Sequencing of Archived MicroRNAs from up to 35-Year-Old Clinical FFPE Specimens

PubMed Central

Loudig, Olivier; Wang, Tao; Ye, Kenny; Lin, Juan; Wang, Yihong; Ramnauth, Andrew; Liu, Christina; Stark, Azadeh; Chitale, Dhananjay; Greenlee, Robert; Multerer, Deborah; Honda, Stacey; Daida, Yihe; Spencer Feigelson, Heather; Glass, Andrew; Couch, Fergus J.; Rohan, Thomas; Ben-Dov, Iddo Z.

2017-01-01

Formalin-fixed paraffin-embedded (FFPE) specimens, when used in conjunction with patient clinical data history, represent an invaluable resource for molecular studies of cancer. Even though nucleic acids extracted from archived FFPE tissues are degraded, their molecular analysis has become possible. In this study, we optimized a laboratory-based next-generation sequencing barcoded cDNA library preparation protocol for analysis of small RNAs recovered from archived FFPE tissues. Using matched fresh and FFPE specimens, we evaluated the robustness and reproducibility of our optimized approach, as well as its applicability to archived clinical specimens stored for up to 35 years. We then evaluated this cDNA library preparation protocol by performing a miRNA expression analysis of archived breast ductal carcinoma in situ (DCIS) specimens, selected for their relation to the risk of subsequent breast cancer development and obtained from six different institutions. Our analyses identified six miRNAs (miR-29a, miR-221, miR-375, miR-184, miR-363, miR-455-5p) differentially expressed between DCIS lesions from women who subsequently developed an invasive breast cancer (cases) and women who did not develop invasive breast cancer within the same time interval (control). Our thorough evaluation and application of this laboratory-based miRNA sequencing analysis indicates that the preparation of small RNA cDNA libraries can reliably be performed on older, archived, clinically-classified specimens. PMID:28335433

Validation of Genotyping-By-Sequencing Analysis in Populations of Tetraploid Alfalfa by 454 Sequencing

PubMed Central

Rocher, Solen; Jean, Martine; Castonguay, Yves; Belzile, François

2015-01-01

Genotyping-by-sequencing (GBS) is a relatively low-cost high throughput genotyping technology based on next generation sequencing and is applicable to orphan species with no reference genome. A combination of genome complexity reduction and multiplexing with DNA barcoding provides a simple and affordable way to resolve allelic variation between plant samples or populations. GBS was performed on ApeKI libraries using DNA from 48 genotypes each of two heterogeneous populations of tetraploid alfalfa (Medicago sativa spp. sativa): the synthetic cultivar Apica (ATF0) and a derived population (ATF5) obtained after five cycles of recurrent selection for superior tolerance to freezing (TF). Nearly 400 million reads were obtained from two lanes of an Illumina HiSeq 2000 sequencer and analyzed with the Universal Network-Enabled Analysis Kit (UNEAK) pipeline designed for species with no reference genome. Following the application of whole dataset-level filters, 11,694 single nucleotide polymorphism (SNP) loci were obtained. About 60% had a significant match on the Medicago truncatula syntenic genome. The accuracy of allelic ratios and genotype calls based on GBS data was directly assessed using 454 sequencing on a subset of SNP loci scored in eight plant samples. Sequencing depth in this study was not sufficient for accurate tetraploid allelic dosage, but reliable genotype calls based on diploid allelic dosage were obtained when using additional quality filtering. Principal Component Analysis of SNP loci in plant samples revealed that a small proportion (<5%) of the genetic variability assessed by GBS is able to differentiate ATF0 and ATF5. Our results confirm that analysis of GBS data using UNEAK is a reliable approach for genome-wide discovery of SNP loci in outcrossed polyploids. PMID:26115486

DNA metabarcoding and microscopic analyses of sea turtles biofilms: Complementary to understand turtle behavior

PubMed Central

Vasselon, Valentin; Ballorain, Katia; Carpentier, Alice; Wetzel, Carlos E.; Ector, Luc; Bouchez, Agnès; Rimet, Frédéric

2018-01-01

Sea turtles are distributed in tropical and subtropical seas worldwide. They play several ecological roles and are considered important indicators of the health of marine ecosystems. Studying epibiotic diatoms living on turtle shells suggestively has great potential in the study of turtle behavior because diatoms are always there. However, diatom identification at the species level is time consuming, requires well-trained specialists, and there is a high probability of finding new taxa growing on turtle shells, which makes identification tricky. An alternative approach based on DNA barcoding and high throughput sequencing (HTS), metabarcoding, has been developed in recent years to identify species at the community level by using a DNA reference library. The suitabilities of morphological and molecular approaches were compared. Diatom assemblages were sampled from seven juvenile green turtles (Chelonia mydas) from Mayotte Island, France. The structures of the epibiotic diatom assemblages differed between both approaches. This resulted in different clustering of the turtles based on their diatom communities. Metabarcoding allowed better discrimination between turtles based on their epibiotic diatom assemblages and put into evidence the presence of a cryptic diatom diversity. Microscopy, for its part, provided more ecological information of sea turtles based on historical bibliographical data and the abundances of ecological guilds of the diatom species present in the samples. This study shows the complementary nature of these two methods for studying turtle behavior. PMID:29659610

DNA-based identification of invasive alien species in relation to Canadian federal policy and law, and the basis of rapid-response management.

PubMed

Thomas, Vernon G; Hanner, Robert H; Borisenko, Alex V

2016-11-01

Managing invasive alien species in Canada requires reliable taxonomic identification as the basis of rapid-response management. This can be challenging, especially when organisms are small and lack morphological diagnostic features. DNA-based techniques, such as DNA barcoding, offer a reliable, rapid, and inexpensive toolkit for taxonomic identification of individual or bulk samples, forensic remains, and even environmental DNA. Well suited for this requirement, they could be more broadly deployed and incorporated into the operating policy and practices of Canadian federal departments and should be authorized under these agencies' articles of law. These include Fisheries and Oceans Canada, Canadian Food Inspection Agency, Transport Canada, Environment Canada, Parks Canada, and Health Canada. These efforts should be harmonized with the appropriate provisions of provincial jurisdictions, for example, the Ontario Invasive Species Act. This approach necessitates that a network of accredited, certified laboratories exists, and that updated DNA reference libraries are readily accessible. Harmonizing this approach is vital among Canadian federal agencies, and between the federal and provincial levels of government. Canadian policy and law must also be harmonized with that of the USA when detecting, and responding to, invasive species in contiguous lands and waters. Creating capacity in legislation for use of DNA-based identifications brings the authority to fund, train, deploy, and certify staff, and to refine further developments in this molecular technology.

DNA metabarcoding and microscopic analyses of sea turtles biofilms: Complementary to understand turtle behavior.

PubMed

Rivera, Sinziana F; Vasselon, Valentin; Ballorain, Katia; Carpentier, Alice; Wetzel, Carlos E; Ector, Luc; Bouchez, Agnès; Rimet, Frédéric

2018-01-01

Sea turtles are distributed in tropical and subtropical seas worldwide. They play several ecological roles and are considered important indicators of the health of marine ecosystems. Studying epibiotic diatoms living on turtle shells suggestively has great potential in the study of turtle behavior because diatoms are always there. However, diatom identification at the species level is time consuming, requires well-trained specialists, and there is a high probability of finding new taxa growing on turtle shells, which makes identification tricky. An alternative approach based on DNA barcoding and high throughput sequencing (HTS), metabarcoding, has been developed in recent years to identify species at the community level by using a DNA reference library. The suitabilities of morphological and molecular approaches were compared. Diatom assemblages were sampled from seven juvenile green turtles (Chelonia mydas) from Mayotte Island, France. The structures of the epibiotic diatom assemblages differed between both approaches. This resulted in different clustering of the turtles based on their diatom communities. Metabarcoding allowed better discrimination between turtles based on their epibiotic diatom assemblages and put into evidence the presence of a cryptic diatom diversity. Microscopy, for its part, provided more ecological information of sea turtles based on historical bibliographical data and the abundances of ecological guilds of the diatom species present in the samples. This study shows the complementary nature of these two methods for studying turtle behavior.

Reading the Complex Skipper Butterfly Fauna of One Tropical Place

PubMed Central

Janzen, Daniel H.; Hallwachs, Winnie; Burns, John M.; Hajibabaei, Mehrdad; Bertrand, Claudia; Hebert, Paul D. N.

2011-01-01

Background An intense, 30-year, ongoing biodiversity inventory of Lepidoptera, together with their food plants and parasitoids, is centered on the rearing of wild-caught caterpillars in the 120,000 terrestrial hectares of dry, rain, and cloud forest of Area de Conservacion Guanacaste (ACG) in northwestern Costa Rica. Since 2003, DNA barcoding of all species has aided their identification and discovery. We summarize the process and results for a large set of the species of two speciose subfamilies of ACG skipper butterflies (Hesperiidae) and emphasize the effectiveness of barcoding these species (which are often difficult and time-consuming to identify). Methodology/Principal Findings Adults are DNA barcoded by the Biodiversity Institute of Ontario, Guelph, Canada; and they are identified by correlating the resulting COI barcode information with more traditional information such as food plant, facies, genitalia, microlocation within ACG, caterpillar traits, etc. This process has found about 303 morphologically defined species of eudamine and pyrgine Hesperiidae breeding in ACG (about 25% of the ACG butterfly fauna) and another 44 units indicated by distinct barcodes (n = 9,094), which may be additional species and therefore may represent as much as a 13% increase. All but the members of one complex can be identified by their DNA barcodes. Conclusions/Significance Addition of DNA barcoding to the methodology greatly improved the inventory, both through faster (hence cheaper) accurate identification of the species that are distinguishable without barcoding, as well as those that require it, and through the revelation of species “hidden” within what have long been viewed as single species. Barcoding increased the recognition of species-level specialization. It would be no more appropriate to ignore barcode data in a species inventory than it would be to ignore adult genitalia variation or caterpillar ecology. PMID:21857895

A regional approach to plant DNA barcoding provides high species resolution of sedges (Carex and Kobresia, Cyperaceae) in the Canadian Arctic Archipelago.

PubMed

Clerc-Blain, Jessica L E; Starr, Julian R; Bull, Roger D; Saarela, Jeffery M

2010-01-01

Previous research on barcoding sedges (Carex) suggested that basic searches within a global barcoding database would probably not resolve more than 60% of the world's some 2000 species. In this study, we take an alternative approach and explore the performance of plant DNA barcoding in the Carex lineage from an explicitly regional perspective. We characterize the utility of a subset of the proposed protein-coding and noncoding plastid barcoding regions (matK, rpoB, rpoC1, rbcL, atpF-atpH, psbK-psbI) for distinguishing species of Carex and Kobresia in the Canadian Arctic Archipelago, a clearly defined eco-geographical region representing 1% of the Earth's landmass. Our results show that matK resolves the greatest number of species of any single-locus (95%), and when combined in a two-locus barcode, it provides 100% species resolution in all but one combination (matK + atpFH) during unweighted pair-group method with arithmetic mean averages (UPGMA) analyses. Noncoding regions were equally or more variable than matK, but as single markers they resolve substantially fewer taxa than matK alone. When difficulties with sequencing and alignment due to microstructural variation in noncoding regions are also considered, our results support other studies in suggesting that protein-coding regions are more practical as barcoding markers. Plastid DNA barcodes are an effective identification tool for species of Carex and Kobresia in the Canadian Arctic Archipelago, a region where the number of co-existing closely related species is limited. We suggest that if a regional approach to plant DNA barcoding was applied on a global scale, it could provide a solution to the generally poor species resolution seen in previous barcoding studies. © 2009 Blackwell Publishing Ltd.

International Society of Human and Animal Mycology (ISHAM)-ITS reference DNA barcoding database--the quality controlled standard tool for routine identification of human and animal pathogenic fungi.

PubMed

Irinyi, Laszlo; Serena, Carolina; Garcia-Hermoso, Dea; Arabatzis, Michael; Desnos-Ollivier, Marie; Vu, Duong; Cardinali, Gianluigi; Arthur, Ian; Normand, Anne-Cécile; Giraldo, Alejandra; da Cunha, Keith Cassia; Sandoval-Denis, Marcelo; Hendrickx, Marijke; Nishikaku, Angela Satie; de Azevedo Melo, Analy Salles; Merseguel, Karina Bellinghausen; Khan, Aziza; Parente Rocha, Juliana Alves; Sampaio, Paula; da Silva Briones, Marcelo Ribeiro; e Ferreira, Renata Carmona; de Medeiros Muniz, Mauro; Castañón-Olivares, Laura Rosio; Estrada-Barcenas, Daniel; Cassagne, Carole; Mary, Charles; Duan, Shu Yao; Kong, Fanrong; Sun, Annie Ying; Zeng, Xianyu; Zhao, Zuotao; Gantois, Nausicaa; Botterel, Françoise; Robbertse, Barbara; Schoch, Conrad; Gams, Walter; Ellis, David; Halliday, Catriona; Chen, Sharon; Sorrell, Tania C; Piarroux, Renaud; Colombo, Arnaldo L; Pais, Célia; de Hoog, Sybren; Zancopé-Oliveira, Rosely Maria; Taylor, Maria Lucia; Toriello, Conchita; de Almeida Soares, Célia Maria; Delhaes, Laurence; Stubbe, Dirk; Dromer, Françoise; Ranque, Stéphane; Guarro, Josep; Cano-Lira, Jose F; Robert, Vincent; Velegraki, Aristea; Meyer, Wieland

2015-05-01

Human and animal fungal pathogens are a growing threat worldwide leading to emerging infections and creating new risks for established ones. There is a growing need for a rapid and accurate identification of pathogens to enable early diagnosis and targeted antifungal therapy. Morphological and biochemical identification methods are time-consuming and require trained experts. Alternatively, molecular methods, such as DNA barcoding, a powerful and easy tool for rapid monophasic identification, offer a practical approach for species identification and less demanding in terms of taxonomical expertise. However, its wide-spread use is still limited by a lack of quality-controlled reference databases and the evolving recognition and definition of new fungal species/complexes. An international consortium of medical mycology laboratories was formed aiming to establish a quality controlled ITS database under the umbrella of the ISHAM working group on "DNA barcoding of human and animal pathogenic fungi." A new database, containing 2800 ITS sequences representing 421 fungal species, providing the medical community with a freely accessible tool at http://www.isham.org/ and http://its.mycologylab.org/ to rapidly and reliably identify most agents of mycoses, was established. The generated sequences included in the new database were used to evaluate the variation and overall utility of the ITS region for the identification of pathogenic fungi at intra-and interspecies level. The average intraspecies variation ranged from 0 to 2.25%. This highlighted selected pathogenic fungal species, such as the dermatophytes and emerging yeast, for which additional molecular methods/genetic markers are required for their reliable identification from clinical and veterinary specimens. © The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A DNA barcode for land plants.

PubMed

2009-08-04

DNA barcoding involves sequencing a standard region of DNA as a tool for species identification. However, there has been no agreement on which region(s) should be used for barcoding land plants. To provide a community recommendation on a standard plant barcode, we have compared the performance of 7 leading candidate plastid DNA regions (atpF-atpH spacer, matK gene, rbcL gene, rpoB gene, rpoC1 gene, psbK-psbI spacer, and trnH-psbA spacer). Based on assessments of recoverability, sequence quality, and levels of species discrimination, we recommend the 2-locus combination of rbcL+matK as the plant barcode. This core 2-locus barcode will provide a universal framework for the routine use of DNA sequence data to identify specimens and contribute toward the discovery of overlooked species of land plants.

A DNA barcode for land plants

PubMed Central

Hollingsworth, Peter M.; Forrest, Laura L.; Spouge, John L.; Hajibabaei, Mehrdad; Ratnasingham, Sujeevan; van der Bank, Michelle; Chase, Mark W.; Cowan, Robyn S.; Erickson, David L.; Fazekas, Aron J.; Graham, Sean W.; James, Karen E.; Kim, Ki-Joong; Kress, W. John; Schneider, Harald; van AlphenStahl, Jonathan; Barrett, Spencer C.H.; van den Berg, Cassio; Bogarin, Diego; Burgess, Kevin S.; Cameron, Kenneth M.; Carine, Mark; Chacón, Juliana; Clark, Alexandra; Clarkson, James J.; Conrad, Ferozah; Devey, Dion S.; Ford, Caroline S.; Hedderson, Terry A.J.; Hollingsworth, Michelle L.; Husband, Brian C.; Kelly, Laura J.; Kesanakurti, Prasad R.; Kim, Jung Sung; Kim, Young-Dong; Lahaye, Renaud; Lee, Hae-Lim; Long, David G.; Madriñán, Santiago; Maurin, Olivier; Meusnier, Isabelle; Newmaster, Steven G.; Park, Chong-Wook; Percy, Diana M.; Petersen, Gitte; Richardson, James E.; Salazar, Gerardo A.; Savolainen, Vincent; Seberg, Ole; Wilkinson, Michael J.; Yi, Dong-Keun; Little, Damon P.

2009-01-01

DNA barcoding involves sequencing a standard region of DNA as a tool for species identification. However, there has been no agreement on which region(s) should be used for barcoding land plants. To provide a community recommendation on a standard plant barcode, we have compared the performance of 7 leading candidate plastid DNA regions (atpF–atpH spacer, matK gene, rbcL gene, rpoB gene, rpoC1 gene, psbK–psbI spacer, and trnH–psbA spacer). Based on assessments of recoverability, sequence quality, and levels of species discrimination, we recommend the 2-locus combination of rbcL+matK as the plant barcode. This core 2-locus barcode will provide a universal framework for the routine use of DNA sequence data to identify specimens and contribute toward the discovery of overlooked species of land plants. PMID:19666622

Validation of the ITS2 Region as a Novel DNA Barcode for Identifying Medicinal Plant Species

PubMed Central

Chen, Shilin; Yao, Hui; Han, Jianping; Liu, Chang; Song, Jingyuan; Shi, Linchun; Zhu, Yingjie; Ma, Xinye; Gao, Ting; Pang, Xiaohui; Luo, Kun; Li, Ying; Li, Xiwen; Jia, Xiaocheng; Lin, Yulin; Leon, Christine

2010-01-01

Background The plant working group of the Consortium for the Barcode of Life recommended the two-locus combination of rbcL + matK as the plant barcode, yet the combination was shown to successfully discriminate among 907 samples from 550 species at the species level with a probability of 72%. The group admits that the two-locus barcode is far from perfect due to the low identification rate, and the search is not over. Methodology/Principal Findings Here, we compared seven candidate DNA barcodes (psbA-trnH, matK, rbcL, rpoC1, ycf5, ITS2, and ITS) from medicinal plant species. Our ranking criteria included PCR amplification efficiency, differential intra- and inter-specific divergences, and the DNA barcoding gap. Our data suggest that the second internal transcribed spacer (ITS2) of nuclear ribosomal DNA represents the most suitable region for DNA barcoding applications. Furthermore, we tested the discrimination ability of ITS2 in more than 6600 plant samples belonging to 4800 species from 753 distinct genera and found that the rate of successful identification with the ITS2 was 92.7% at the species level. Conclusions The ITS2 region can be potentially used as a standard DNA barcode to identify medicinal plants and their closely related species. We also propose that ITS2 can serve as a novel universal barcode for the identification of a broader range of plant taxa. PMID:20062805

Multilocus inference of species trees and DNA barcoding.

PubMed

Mallo, Diego; Posada, David

2016-09-05

The unprecedented amount of data resulting from next-generation sequencing has opened a new era in phylogenetic estimation. Although large datasets should, in theory, increase phylogenetic resolution, massive, multilocus datasets have uncovered a great deal of phylogenetic incongruence among different genomic regions, due both to stochastic error and to the action of different evolutionary process such as incomplete lineage sorting, gene duplication and loss and horizontal gene transfer. This incongruence violates one of the fundamental assumptions of the DNA barcoding approach, which assumes that gene history and species history are identical. In this review, we explain some of the most important challenges we will have to face to reconstruct the history of species, and the advantages and disadvantages of different strategies for the phylogenetic analysis of multilocus data. In particular, we describe the evolutionary events that can generate species tree-gene tree discordance, compare the most popular methods for species tree reconstruction, highlight the challenges we need to face when using them and discuss their potential utility in barcoding. Current barcoding methods sacrifice a great amount of statistical power by only considering one locus, and a transition to multilocus barcodes would not only improve current barcoding methods, but also facilitate an eventual transition to species-tree-based barcoding strategies, which could better accommodate scenarios where the barcode gap is too small or inexistent.This article is part of the themed issue 'From DNA barcodes to biomes'. © 2016 The Authors.

Decision Tree Algorithm-Generated Single-Nucleotide Polymorphism Barcodes of rbcL Genes for 38 Brassicaceae Species Tagging.

PubMed

Yang, Cheng-Hong; Wu, Kuo-Chuan; Chuang, Li-Yeh; Chang, Hsueh-Wei

2018-01-01

DNA barcode sequences are accumulating in large data sets. A barcode is generally a sequence larger than 1000 base pairs and generates a computational burden. Although the DNA barcode was originally envisioned as straightforward species tags, the identification usage of barcode sequences is rarely emphasized currently. Single-nucleotide polymorphism (SNP) association studies provide us an idea that the SNPs may be the ideal target of feature selection to discriminate between different species. We hypothesize that SNP-based barcodes may be more effective than the full length of DNA barcode sequences for species discrimination. To address this issue, we tested a r ibulose diphosphate carboxylase ( rbcL ) S NP b arcoding (RSB) strategy using a decision tree algorithm. After alignment and trimming, 31 SNPs were discovered in the rbcL sequences from 38 Brassicaceae plant species. In the decision tree construction, these SNPs were computed to set up the decision rule to assign the sequences into 2 groups level by level. After algorithm processing, 37 nodes and 31 loci were required for discriminating 38 species. Finally, the sequence tags consisting of 31 rbcL SNP barcodes were identified for discriminating 38 Brassicaceae species based on the decision tree-selected SNP pattern using RSB method. Taken together, this study provides the rational that the SNP aspect of DNA barcode for rbcL gene is a useful and effective sequence for tagging 38 Brassicaceae species.

Supervisory Rotation: Impact on an Academic Library Reference Staff.

ERIC Educational Resources Information Center

Perdue, Bob; Piotrowski, Chris

1986-01-01

Presents results of managerial rotation program (librarians take turns on two-year cycle serving as department head) in medium-sized academic library reference department. An assessment of both the advantages and disadvantages of such a plan examines impact of this innovative and collegial management technique on staff, library, and patrons. (7…

Digital Library and Digital Reference Service: Integration and Mutual Complementarity

ERIC Educational Resources Information Center

Liu, Jia

2008-01-01

Both the digital library and the digital reference service were invented and have been developed under the networked environment. Among their intersections, the fundamental thing is their symbiotic interest--serving the user in a more efficient way. The article starts by discussing the digital library and its service and the digital reference…

User Education in the Academic Library: Media Methods for Reference Work. An Annotated Bibliography. Updated.

ERIC Educational Resources Information Center

Wharton, Sika

This annotated bibliography focuses on academic library usage of audiovisual (AV) methods of instruction, particularly for the enhancement of the reference teaching function. The bibliography's objectives are as follows: to identify current trends with regard to AV methods in library orientation and bibliographic instruction; to isolate instances…

Genetic barcodes

DOEpatents

Weier, Heinz -Ulrich G

2015-08-04

Herein are described multicolor FISH probe sets termed "genetic barcodes" targeting several cancer or disease-related loci to assess gene rearrangements and copy number changes in tumor cells. Two, three or more different fluorophores are used to detect the genetic barcode sections thus permitting unique labeling and multilocus analysis in individual cell nuclei. Gene specific barcodes can be generated and combined to provide both numerical and structural genetic information for these and other pertinent disease associated genes.

Accuracy and time requirements of a bar-code inventory system for medical supplies.

PubMed

Hanson, L B; Weinswig, M H; De Muth, J E

1988-02-01

The effects of implementing a bar-code system for issuing medical supplies to nursing units at a university teaching hospital were evaluated. Data on the time required to issue medical supplies to three nursing units at a 480-bed, tertiary-care teaching hospital were collected (1) before the bar-code system was implemented (i.e., when the manual system was in use), (2) one month after implementation, and (3) four months after implementation. At the same times, the accuracy of the central supply perpetual inventory was monitored using 15 selected items. One-way analysis of variance tests were done to determine any significant differences between the bar-code and manual systems. Using the bar-code system took longer than using the manual system because of a significant difference in the time required for order entry into the computer. Multiple-use requirements of the central supply computer system made entering bar-code data a much slower process. There was, however, a significant improvement in the accuracy of the perpetual inventory. Using the bar-code system for issuing medical supplies to the nursing units takes longer than using the manual system. However, the accuracy of the perpetual inventory was significantly improved with the implementation of the bar-code system.

A smartphone-readable barcode assay for the detection and quantitation of pesticide residues.

PubMed

Guo, Juan; Wong, Jessica X H; Cui, Caie; Li, Xiaochun; Yu, Hua-Zhong

2015-08-21

In this paper, we present a smartphone-readable barcode assay for the qualitative detection of methyl parathion residues, a toxic organophosphorus pesticide that is popularly used in agriculture worldwide. The detection principle is based on the irreversible inhibition of the enzymatic activity of acetylcholinesterase (AchE) by methyl parathion; AchE catalytically hydrolyzes acetylthiocholine iodine to thiocholine that in turn dissociates dithiobis-nitrobenzoate to produce a yellow product (deprotonated thio-nitrobenzoate). The yellow intensity of the product was confirmed to be inversely dependent on the concentration of the pesticide. We have designed a barcode-formatted assay chip by using a PDMS (polydimethylsiloxane) channel plate (as the reaction reservoir), situated under a printed partial barcode, to complete the whole barcode such that it can be directly read by a barcode scanning app installed on a smartphone. The app is able to qualitatively present the result of the pesticide test; the absence or a low concentration of methyl parathion results in the barcode reading as "-", identifying the test as negative for pesticides. Upon obtaining a positive result (the app reads a "+" character), the captured image can be further analyzed to quantitate the methyl parathion concentration in the sample. Besides the portability and simplicity, this mobile-app based colorimetric barcode assay compares favorably with the standard spectrophotometric method.

CD-ROM Stress.

ERIC Educational Resources Information Center

Bunge, Charles A.

1991-01-01

Discussion of stress in library reference departments focuses on stress caused by CD-ROM reference tools. Topics discussed include work overload; nonreference duties; patron attitudes and behavior; staff attitudes; the need for proper staff training; and the need for library administrators to be sensitive to reference staff needs. (LRW)

Changing Roles for References Librarians.

ERIC Educational Resources Information Center

Kelly, Julia; Robbins, Kathryn

1996-01-01

Discusses the future outlook for reference librarians, with topics including: "Technology as the Source of Change"; "Impact of the Internet"; "Defining the Virtual Library"; "Rethinking Reference"; "Out of the Library and into the Streets"; "Asking Users About Their Needs"; "Standardization and Artificial Intelligence"; "The Financial Future"; and…

Online Reference Service--How to Begin: A Selected Bibliography.

ERIC Educational Resources Information Center

Shroder, Emelie J., Ed.

1982-01-01

Materials in this bibliography were selected and recommended by members of the Use of Machine-Assisted Reference in Public Libraries Committee, Reference and Adult Services Division, American Library Association. Topics include: financial aspects, equipment and communications considerations, comparing databases and database systems, advertising…

Reference Collection Development Policy. Penfield Library, State University of New York College at Oswego.

ERIC Educational Resources Information Center

Judd, Blanche; And Others

Intended for use by the head of reference and subject-area bibliographers at Penfield Library, this document provides guidelines for the selection and evaluation of titles for the 25,000 volume reference collection at this primarily undergraduate institution. The reference collection also supports the general information needs of both the college…

The University of Texas at Arlington's Virtual Reference Service: An Evaluation by the Reference Staff

ERIC Educational Resources Information Center

Casebier, Katherine D.

2006-01-01

The University of Texas at Arlington's Library began using an online chat reference in 2002. The service, called Collaborative Digital Reference Service, later became "Ask a Librarian." Slightly over one year later, the library joined the University of Texas System's "Ask a Librarian" service. Both services are powered by…

We Jumped on the Live Reference Band Wagon, and We Love the Ride!

ERIC Educational Resources Information Center

Schaake, Glenda; Sathan, Eleanor

2003-01-01

Memorial Hall Library in Andover, Massachusetts wanted to offer live reference service online but with limited resources, they couldn't do it alone. The 24/7 Reference cooperative program administered by the California State Library required Memorial Hall's librarians to monitor only 10 hours a week in return for live reference coverage. Memorial…

Dissecting a complex chemical stress: chemogenomic profiling of plant hydrolysates

PubMed Central

Skerker, Jeffrey M; Leon, Dacia; Price, Morgan N; Mar, Jordan S; Tarjan, Daniel R; Wetmore, Kelly M; Deutschbauer, Adam M; Baumohl, Jason K; Bauer, Stefan; Ibáñez, Ana B; Mitchell, Valerie D; Wu, Cindy H; Hu, Ping; Hazen, Terry; Arkin, Adam P

2013-01-01

The efficient production of biofuels from cellulosic feedstocks will require the efficient fermentation of the sugars in hydrolyzed plant material. Unfortunately, plant hydrolysates also contain many compounds that inhibit microbial growth and fermentation. We used DNA-barcoded mutant libraries to identify genes that are important for hydrolysate tolerance in both Zymomonas mobilis (44 genes) and Saccharomyces cerevisiae (99 genes). Overexpression of a Z. mobilis tolerance gene of unknown function (ZMO1875) improved its specific ethanol productivity 2.4-fold in the presence of miscanthus hydrolysate. However, a mixture of 37 hydrolysate-derived inhibitors was not sufficient to explain the fitness profile of plant hydrolysate. To deconstruct the fitness profile of hydrolysate, we profiled the 37 inhibitors against a library of Z. mobilis mutants and we modeled fitness in hydrolysate as a mixture of fitness in its components. By examining outliers in this model, we identified methylglyoxal as a previously unknown component of hydrolysate. Our work provides a general strategy to dissect how microbes respond to a complex chemical stress and should enable further engineering of hydrolysate tolerance. PMID:23774757

The Allama Iqbal Open University Library.

ERIC Educational Resources Information Center

Ul-Hassan, Mahmud

1984-01-01

This profile of an open university library system in Pakistan covers its primary objective; functional organization; library collection and collection development; staffing, budgets and expenditures; library services (users, technical, reference); departmental libraries (on-campus branch, regional); and the library building. A list of 16 library…

Submicrometre geometrically encoded fluorescent barcodes self-assembled from DNA

NASA Astrophysics Data System (ADS)

Lin, Chenxiang; Jungmann, Ralf; Leifer, Andrew M.; Li, Chao; Levner, Daniel; Church, George M.; Shih, William M.; Yin, Peng

2012-10-01

The identification and differentiation of a large number of distinct molecular species with high temporal and spatial resolution is a major challenge in biomedical science. Fluorescence microscopy is a powerful tool, but its multiplexing ability is limited by the number of spectrally distinguishable fluorophores. Here, we used (deoxy)ribonucleic acid (DNA)-origami technology to construct submicrometre nanorods that act as fluorescent barcodes. We demonstrate that spatial control over the positioning of fluorophores on the surface of a stiff DNA nanorod can produce 216 distinct barcodes that can be decoded unambiguously using epifluorescence or total internal reflection fluorescence microscopy. Barcodes with higher spatial information density were demonstrated via the construction of super-resolution barcodes with features spaced by ˜40 nm. One species of the barcodes was used to tag yeast surface receptors, which suggests their potential applications as in situ imaging probes for diverse biomolecular and cellular entities in their native environments.

DNA barcoding of odonates from the Upper Plata basin: Database creation and genetic diversity estimation.

PubMed

Koroiva, Ricardo; Pepinelli, Mateus; Rodrigues, Marciel Elio; Roque, Fabio de Oliveira; Lorenz-Lemke, Aline Pedroso; Kvist, Sebastian

2017-01-01

We present a DNA barcoding study of Neotropical odonates from the Upper Plata basin, Brazil. A total of 38 species were collected in a transition region of "Cerrado" and Atlantic Forest, both regarded as biological hotspots, and 130 cytochrome c oxidase subunit I (COI) barcodes were generated for the collected specimens. The distinct gap between intraspecific (0-2%) and interspecific variation (15% and above) in COI, and resulting separation of Barcode Index Numbers (BIN), allowed for successful identification of specimens in 94% of cases. The 6% fail rate was due to a shared BIN between two separate nominal species. DNA barcoding, based on COI, thus seems to be a reliable and efficient tool for identifying Neotropical odonate specimens down to the species level. These results underscore the utility of DNA barcoding to aid specimen identification in diverse biological hotspots, areas that require urgent action regarding taxonomic surveys and biodiversity conservation.

Scanning for safety: an integrated approach to improved bar-code medication administration.

PubMed

Early, Cynde; Riha, Chris; Martin, Jennifer; Lowdon, Karen W; Harvey, Ellen M

2011-03-01

This is a review of lessons learned in the postimplementation evaluation of a bar-code medication administration technology implemented at a major tertiary-care hospital in 2001. In 2006, with a bar-code medication administration scan compliance rate of 82%, a near-miss sentinel event prompted review of this technology as part of an institutional recommitment to a "culture of safety." Multifaceted problems with bar-code medication administration created an environment of circumventing safeguards as demonstrated by an increase in manual overrides to ensure timely medication administration. A multiprofessional team composed of nursing, pharmacy, human resources, quality, and technical services formalized. Each step in the bar-code medication administration process was reviewed. Technology, process, and educational solutions were identified and implemented systematically. Overall compliance with bar-code medication administration rose from 82% to 97%, which resulted in a calculated cost avoidance of more than $2.8 million during this time frame of the project.

Sub-micrometer Geometrically Encoded Fluorescent Barcodes Self-Assembled from DNA

PubMed Central

Lin, Chenxiang; Jungmann, Ralf; Leifer, Andrew M.; Li, Chao; Levner, Daniel; Church, George M.; Shih, William M.; Yin, Peng

2012-01-01

The identification and differentiation of a large number of distinct molecular species with high temporal and spatial resolution is a major challenge in biomedical science. Fluorescence microscopy is a powerful tool, but its multiplexing ability is limited by the number of spectrally distinguishable fluorophores. Here we use DNA-origami technology to construct sub-micrometer nanorods that act as fluorescent barcodes. We demonstrate that spatial control over the positioning of fluorophores on the surface of a stiff DNA nanorod can produce 216 distinct barcodes that can be unambiguously decoded using epifluorescence or total internal reflection fluorescence (TIRF) microscopy. Barcodes with higher spatial information density were demonstrated via the construction of super-resolution barcodes with features spaced by ~40 nm. One species of the barcodes was used to tag yeast surface receptors, suggesting their potential applications as in situ imaging probes for diverse biomolecular and cellular entities in their native environments. PMID:23000997

Gold Nanoparticles-Based Barcode Analysis for Detection of Norepinephrine.

PubMed

An, Jeung Hee; Lee, Kwon-Jai; Choi, Jeong-Woo

2016-02-01

Nanotechnology-based bio-barcode amplification analysis offers an innovative approach for detecting neurotransmitters. We evaluated the efficacy of this method for detecting norepinephrine in normal and oxidative-stress damaged dopaminergic cells. Our approach use a combination of DNA barcodes and bead-based immunoassays for detecting neurotransmitters with surface-enhanced Raman spectroscopy (SERS), and provides polymerase chain reaction (PCR)-like sensitivity. This method relies on magnetic Dynabeads containing antibodies and nanoparticles that are loaded both with DNA barcords and with antibodies that can sandwich the target protein captured by the Dynabead-bound antibodies. The aggregate sandwich structures are magnetically separated from the solution and treated to remove the conjugated barcode DNA. The DNA barcodes are then identified by SERS and PCR analysis. The concentration of norepinephrine in dopaminergic cells can be readily detected using the bio-barcode assay, which is a rapid, high-throughput screening tool for detecting neurotransmitters.

University Library Online Reference Service Program Plan, 1986/87.

ERIC Educational Resources Information Center

Koga, James S.

This program plan for online reference service--the individualized assistance provided to a library patron using an online system--at California State Polytechnic University, Pomona, covers the areas of funding, eligibility for online services, search request eligibility, database eligibility, management of online services, reference faculty…

Outstanding Reference Sources: The 1994 Selection of Recent Titles.

ERIC Educational Resources Information Center

Luchsinger, Dale F.

1994-01-01

Presents an annotated bibliography of 37 outstanding reference sources published in 1993 for small to medium-sized public and academic libraries as selected by the American Library Association's Reference Services Committee. Categories include culture and civilization, biography, general, language and literature, nature, law, religion, technology,…

CD-ROM + Fax = Shared Reference Resources.

ERIC Educational Resources Information Center

Fitzwater, Diana; Fradkin, Bernard

1988-01-01

Describes the Reference by GammaFax Project, which joined nine area libraries to provide cooperative reference access to optical disk databases. The configuration of disk players, microcomputers, and facsimile equipment used by the libraries is explained, and the improvements in cost effectiveness, provision of service, and librarian expertise are…

Going Prime Time with Live Chat Reference.

ERIC Educational Resources Information Center

Hoag, Tara J.; Cichanowicz, Edana McCaffrey

2001-01-01

Describes the development of the Suffolk Cooperative Library System's live, online chat reference service, a pilot project for public libraries in Suffolk County (New York). Topics include chat software selection; a virtual reference collection; marketing; funding; staffing; evaluation; expanded hours of service; email; and extracting data from…

HIV/AIDS reference questions in an AIDS service organization special library.

PubMed

Deevey, Sharon; Behring, Michael

2005-01-01

Librarians in many venues may anticipate a wide range of reference questions related to HIV and AIDS. Information on HIV/ AIDS is now available in medical, academic, and public libraries and on the Internet, and ranges from the most complex science to the most private disclosures about personal behavior. In this article, the 913 reference questions asked between May 2002 and August 2004 in a special library in a mid-western community-based AIDS service organization are described and analyzed.

The Region 4 collaborative virtual reference project.

PubMed

Parker, Sandi K; Johnson, E Diane

2003-01-01

In May 2002, the Denison Memorial Library at the University of Colorado Health Sciences Center and the J. Otto Lottes Health Sciences Library at the University of Missouri-Columbia, with funding from the National Network of Libraries of Medicine-Midcontinental Region, embarked on a collaborative, real-time reference project using the 24/7 Reference, Inc., software package. This paper describes how the project was conceived, and includes details on the service hours, staffing, training, marketing, lessons learned, and future plans for the service.

Strategies for Achieving High Sequencing Accuracy for Low Diversity Samples and Avoiding Sample Bleeding Using Illumina Platform

PubMed Central

Mitra, Abhishek; Skrzypczak, Magdalena; Ginalski, Krzysztof; Rowicka, Maga

2015-01-01

Sequencing microRNA, reduced representation sequencing, Hi-C technology and any method requiring the use of in-house barcodes result in sequencing libraries with low initial sequence diversity. Sequencing such data on the Illumina platform typically produces low quality data due to the limitations of the Illumina cluster calling algorithm. Moreover, even in the case of diverse samples, these limitations are causing substantial inaccuracies in multiplexed sample assignment (sample bleeding). Such inaccuracies are unacceptable in clinical applications, and in some other fields (e.g. detection of rare variants). Here, we discuss how both problems with quality of low-diversity samples and sample bleeding are caused by incorrect detection of clusters on the flowcell during initial sequencing cycles. We propose simple software modifications (Long Template Protocol) that overcome this problem. We present experimental results showing that our Long Template Protocol remarkably increases data quality for low diversity samples, as compared with the standard analysis protocol; it also substantially reduces sample bleeding for all samples. For comprehensiveness, we also discuss and compare experimental results from alternative approaches to sequencing low diversity samples. First, we discuss how the low diversity problem, if caused by barcodes, can be avoided altogether at the barcode design stage. Second and third, we present modified guidelines, which are more stringent than the manufacturer’s, for mixing low diversity samples with diverse samples and lowering cluster density, which in our experience consistently produces high quality data from low diversity samples. Fourth and fifth, we present rescue strategies that can be applied when sequencing results in low quality data and when there is no more biological material available. In such cases, we propose that the flowcell be re-hybridized and sequenced again using our Long Template Protocol. Alternatively, we discuss how analysis can be repeated from saved sequencing images using the Long Template Protocol to increase accuracy. PMID:25860802

Doing Virtual Reference along with Everything Else

ERIC Educational Resources Information Center

Garlish, Betsy Harper

2009-01-01

Virtual reference is a service that makes particular sense in a state like Montana. The fourth-largest state by geographic area but 44th by population in the U.S., it has about one library per 11,392 residents. Montana's libraries, including the libraries of Montana Tech of the University of Montana, where the author currently serves as a…

The Teaching of Reference in American Library Schools. Historical Paper 4

ERIC Educational Resources Information Center

Cheney, Frances Neel

2015-01-01

In this article, the author proposes to present a superficial view of what is being taught in the 32 American Library Association accredited graduate library schools. She briefly discusses and answers the following three questions: (1) Who is teaching reference?; (2) What is being taught?; and (3) How is it being taught? [For the commentary on…