Genetic Patterns in European Geometrid Moths Revealed by the Barcode Index Number (BIN) System

Background

The geometrid moths of Europe are one of the best investigated insect groups in traditional taxonomy making them an ideal model group to test the accuracy of the Barcode Index Number (BIN) system of BOLD (Barcode of Life Datasystems), a method that supports automated, rapid species delineation and identification.

Methodology/Principal Findings

This study provides a DNA barcode library for 219 of the 249 European geometrid moth species (88%) in five selected subfamilies. The data set includes COI sequences for 2130 specimens. Most species (93%) were found to possess diagnostic barcode sequences at the European level while only three species pairs (3%) were genetically indistinguishable in areas of sympatry. As a consequence, 97% of the European species we examined were unequivocally discriminated by barcodes within their natural areas of distribution. We found a 1:1 correspondence between BINs and traditionally recognized species for 67% of these species. Another 17% of the species (15 pairs, three triads) shared BINs, while specimens from the remaining species (18%) were divided among two or more BINs. Five of these species are mixtures, both sharing and splitting BINs. For 82% of the species with two or more BINs, the genetic splits involved allopatric populations, many of which have previously been hypothesized to represent distinct species or subspecies.

Conclusions/Significance

This study confirms the effectiveness of DNA barcoding as a tool for species identification and illustrates the potential of the BIN system to characterize formal genetic units independently of an existing classification. This suggests the system can be used to efficiently assess the biodiversity of large, poorly known assemblages of organisms. For the moths examined in this study, cases of discordance between traditionally recognized species and BINs arose from several causes including overlooked species, synonymy, and cases where DNA barcodes revealed regional variation of uncertain taxonomic significance.     

DNA Barcoding for Species Assignment: The Case of Mediterranean Marine Fishes

Background

DNA barcoding enhances the prospects for species-level identifications globally using a standardized and authenticated DNA-based approach. Reference libraries comprising validated DNA barcodes (COI) constitute robust datasets for testing query sequences, providing considerable utility to identify marine fish and other organisms. Here we test the feasibility of using DNA barcoding to assign species to tissue samples from fish collected in the central Mediterranean Sea, a major contributor to the European marine ichthyofaunal diversity.

Methodology/Principal Findings

A dataset of 1278 DNA barcodes, representing 218 marine fish species, was used to test the utility of DNA barcodes to assign species from query sequences. We tested query sequences against 1) a reference library of ranked DNA barcodes from the neighbouring North East Atlantic, and 2) the public databases BOLD and GenBank. In the first case, a reference library comprising DNA barcodes with reliability grades for 146 fish species was used as diagnostic dataset to screen 486 query DNA sequences from fish specimens collected in the central basin of the Mediterranean Sea. Of all query sequences suitable for comparisons 98% were unambiguously confirmed through complete match with reference DNA barcodes. In the second case, it was possible to assign species to 83% (BOLD-IDS) and 72% (GenBank) of the sequences from the Mediterranean. Relatively high intraspecific genetic distances were found in 7 species (2.2%–18.74%), most of them of high commercial relevance, suggesting possible cryptic species.

Conclusion/Significance

We emphasize the discriminatory power of COI barcodes and their application to cases requiring species level resolution starting from query sequences. Results highlight the value of public reference libraries of reliability grade-annotated DNA barcodes, to identify species from different geographical origins. The ability to assign species with high precision from DNA samples of disparate quality and origin has major utility in several fields, from fisheries and conservation programs to control of fish products authenticity.     

The Role of DNA Barcodes in Understanding and Conservation of Mammal Diversity in Southeast Asia

Background

Southeast Asia is recognized as a region of very high biodiversity, much of which is currently at risk due to habitat loss and other threats. However, many aspects of this diversity, even for relatively well-known groups such as mammals, are poorly known, limiting ability to develop conservation plans. This study examines the value of DNA barcodes, sequences of the mitochondrial COI gene, to enhance understanding of mammalian diversity in the region and hence to aid conservation planning.

Methodology and Principal Findings

DNA barcodes were obtained from nearly 1900 specimens representing 165 recognized species of bats. All morphologically or acoustically distinct species, based on classical taxonomy, could be discriminated with DNA barcodes except four closely allied species pairs. Many currently recognized species contained multiple barcode lineages, often with deep divergence suggesting unrecognized species. In addition, most widespread species showed substantial genetic differentiation across their distributions. Our results suggest that mammal species richness within the region may be underestimated by at least 50%, and there are higher levels of endemism and greater intra-specific population structure than previously recognized.

Conclusions

DNA barcodes can aid conservation and research by assisting field workers in identifying species, by helping taxonomists determine species groups needing more detailed analysis, and by facilitating the recognition of the appropriate units and scales for conservation planning.     

Barcoding bugs: DNA-based identification of the true bugs (Insecta: Hemiptera: Heteroptera)

Background

DNA barcoding, the analysis of sequence variation in the 5′ region of the mitochondrial cytochrome c oxidase I (COI) gene, has been shown to provide an efficient method for the identification of species in a wide range of animal taxa. In order to assess the effectiveness of barcodes in the discrimination of Heteroptera, we examined 344 species belonging to 178 genera, drawn from specimens in the Canadian National Collection of Insects.

Methodology/Principal Findings

Analysis of the COI gene revealed less than 2% intra-specific divergence in 90% of the taxa examined, while minimum interspecific distances exceeded 3% in 77% of congeneric species pairs. Instances where barcodes fail to distinguish species represented clusters of morphologically similar species, except one case of barcode identity between species in different genera. Several instances of deep intraspecific divergence were detected suggesting possible cryptic species.

Conclusions/Significance

Although this analysis encompasses 0.8% of the described global fauna, our results indicate that DNA barcodes will aid the identification of Heteroptera. This advance will be useful in pest management, regulatory and environmental applications and will also reveal species that require further taxonomic research.     

Environmental barcoding reveals massive dinoflagellate diversity in marine environments

Background

Dinoflagellates are an ecologically important group of protists with important functions as primary producers, coral symbionts and in toxic red tides. Although widely studied, the natural diversity of dinoflagellates is not well known. DNA barcoding has been utilized successfully for many protist groups. We used this approach to systematically sample known “species”, as a reference to measure the natural diversity in three marine environments.

Methodology/Principal Findings

In this study, we assembled a large cytochrome c oxidase 1 (COI) barcode database from 8 public algal culture collections plus 3 private collections worldwide resulting in 336 individual barcodes linked to specific cultures. We demonstrate that COI can identify to the species level in 15 dinoflagellate genera, generally in agreement with existing species names. Exceptions were found in species belonging to genera that were generally already known to be taxonomically challenging, such as Alexandrium or Symbiodinium. Using this barcode database as a baseline for cultured dinoflagellate diversity, we investigated the natural diversity in three diverse marine environments (Northeast Pacific, Northwest Atlantic, and Caribbean), including an evaluation of single-cell barcoding to identify uncultivated groups. From all three environments, the great majority of barcodes were not represented by any known cultured dinoflagellate, and we also observed an explosion in the diversity of genera that previously contained a modest number of known species, belonging to Kareniaceae. In total, 91.5% of non-identical environmental barcodes represent distinct species, but only 51 out of 603 unique environmental barcodes could be linked to cultured species using a conservative cut-off based on distances between cultured species.

Conclusions/Significance

COI barcoding was successful in identifying species from 70% of cultured genera. When applied to environmental samples, it revealed a massive amount of natural diversity in dinoflagellates. This highlights the extent to which we underestimate microbial diversity in the environment.     

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

Background

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.

Methodology/Principal Findings

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.

Conclusions/Significance

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.     

Assessing the value of DNA barcodes for molecular phylogenetics: effect of increased taxon sampling in lepidoptera

Background

A common perception is that DNA barcode datamatrices have limited phylogenetic signal due to the small number of characters available per taxon. However, another school of thought suggests that the massively increased taxon sampling afforded through the use of DNA barcodes may considerably increase the phylogenetic signal present in a datamatrix. Here I test this hypothesis using a large dataset of macrolepidopteran DNA barcodes.

Methodology/Principal Findings

Taxon sampling was systematically increased in datamatrices containing macrolepidopteran DNA barcodes. Sixteen family groups were designated as concordance groups and two quantitative measures; the taxon consistency index and the taxon retention index, were used to assess any changes in phylogenetic signal as a result of the increase in taxon sampling. DNA barcodes alone, even with maximal taxon sampling (500 species per family), were not sufficient to reconstruct monophyly of families and increased taxon sampling generally increased the number of clades formed per family. However, the scores indicated a similar level of taxon retention (species from a family clustering together) in the cladograms as the number of species included in the datamatrix was increased, suggesting substantial phylogenetic signal below the ‘family’ branch.

Conclusions/Significance

The development of supermatrix, supertree or constrained tree approaches could enable the exploitation of the massive taxon sampling afforded through DNA barcodes for phylogenetics, connecting the twigs resolved by barcodes to the deep branches resolved through phylogenomics.     

Reading the complex skipper butterfly fauna of one tropical place

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.     

DNA barcoding the geometrid fauna of Bavaria (Lepidoptera): successes, surprises, and questions

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.     

A ranking system for reference libraries of DNA barcodes: application to marine fish species from Portugal

Background

The increasing availability of reference libraries of DNA barcodes (RLDB) offers the opportunity to the screen the level of consistency in DNA barcode data among libraries, in order to detect possible disagreements generated from taxonomic uncertainty or operational shortcomings. We propose a ranking system to attribute a confidence level to species identifications associated with DNA barcode records from a RLDB. Here we apply the proposed ranking system to a newly generated RLDB for marine fish of Portugal.

Methodology/Principal Findings

Specimens (n = 659) representing 102 marine fish species were collected along the continental shelf of Portugal, morphologically identified and archived in a museum collection. Samples were sequenced at the barcode region of the cytochrome oxidase subunit I gene (COI-5P). Resultant DNA barcodes had average intra-specific and inter-specific Kimura-2-parameter distances (0.32% and 8.84%, respectively) within the range usually observed for marine fishes. All specimens were ranked in five different levels (A–E), according to the reliability of the match between their species identification and the respective diagnostic DNA barcodes. Grades A to E were attributed upon submission of individual specimen sequences to BOLD-IDS and inspection of the clustering pattern in the NJ tree generated. Overall, our study resulted in 73.5% of unambiguous species IDs (grade A), 7.8% taxonomically congruent barcode clusters within our dataset, but awaiting external confirmation (grade B), and 18.7% of species identifications with lower levels of reliability (grades C/E).

Conclusion/Significance

We highlight the importance of implementing a system to rank barcode records in RLDB, in order to flag taxa in need of taxonomic revision, or reduce ambiguities of discordant data. With increasing DNA barcode records publicly available, this cross-validation system would provide a metric of relative accuracy of barcodes, while enabling the continuous revision and annotation required in taxonomic work.     

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

Background

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.

Methodology/Principal Findings

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.

Conclusion/Significance

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.     

Barcoding Poplars (Populus L.) from Western China

Background

Populus is an ecologically and economically important genus of trees, but distinguishing between wild species is relatively difficult due to extensive interspecific hybridization and introgression, and the high level of intraspecific morphological variation. The DNA barcoding approach is a potential solution to this problem.

Methodology/Principal Findings

Here, we tested the discrimination power of five chloroplast barcodes and one nuclear barcode (ITS) among 95 trees that represent 21 Populus species from western China. Among all single barcode candidates, the discrimination power is highest for the nuclear ITS, progressively lower for chloroplast barcodes matK (M), trnG-psbK (G) and psbK-psbI (P), and trnH-psbA (H) and rbcL (R); the discrimination efficiency of the nuclear ITS (I) is also higher than any two-, three-, or even the five-locus combination of chloroplast barcodes. Among the five combinations of a single chloroplast barcode plus the nuclear ITS, H+I and P+I differentiated the highest and lowest portion of species, respectively. The highest discrimination rate for the barcodes or barcode combinations examined here is 55.0% (H+I), and usually discrimination failures occurred among species from sympatric or parapatric areas.

Conclusions/Significance

In this case study, we showed that when discriminating Populus species from western China, the nuclear ITS region represents a more promising barcode than any maternally inherited chloroplast region or combination of chloroplast regions. Meanwhile, combining the ITS region with chloroplast regions may improve the barcoding success rate and assist in detecting recent interspecific hybridizations. Failure to discriminate among several groups of Populus species from sympatric or parapatric areas may have been the result of incomplete lineage sorting, frequent interspecific hybridizations and introgressions. We agree with a previous proposal for constructing a tiered barcoding system in plants, especially for taxonomic groups that have complex evolutionary histories (e.g. Populus).     

Patterns of DNA Barcode Variation in Canadian Marine Molluscs

Background

Molluscs are the most diverse marine phylum and this high diversity has resulted in considerable taxonomic problems. Because the number of species in Canadian oceans remains uncertain, there is a need to incorporate molecular methods into species identifications. A 648 base pair segment of the cytochrome c oxidase subunit I gene has proven useful for the identification and discovery of species in many animal lineages. While the utility of DNA barcoding in molluscs has been demonstrated in other studies, this is the first effort to construct a DNA barcode registry for marine molluscs across such a large geographic area.

Methodology/Principal Findings

This study examines patterns of DNA barcode variation in 227 species of Canadian marine molluscs. Intraspecific sequence divergences ranged from 0–26.4% and a barcode gap existed for most taxa. Eleven cases of relatively deep (>2%) intraspecific divergence were detected, suggesting the possible presence of overlooked species. Structural variation was detected in COI with indels found in 37 species, mostly bivalves. Some indels were present in divergent lineages, primarily in the region of the first external loop, suggesting certain areas are hotspots for change. Lastly, mean GC content varied substantially among orders (24.5%–46.5%), and showed a significant positive correlation with nearest neighbour distances.

Conclusions/Significance

DNA barcoding is an effective tool for the identification of Canadian marine molluscs and for revealing possible cases of overlooked species. Some species with deep intraspecific divergence showed a biogeographic partition between lineages on the Atlantic, Arctic and Pacific coasts, suggesting the role of Pleistocene glaciations in the subdivision of their populations. Indels were prevalent in the barcode region of the COI gene in bivalves and gastropods. This study highlights the efficacy of DNA barcoding for providing insights into sequence variation across a broad taxonomic group on a large geographic scale.     

Molecular approach to the identification of fish in the South China Sea

Background

DNA barcoding is one means of establishing a rapid, accurate, and cost-effective system for the identification of species. It involves the use of short, standard gene targets to create sequence profiles of known species against sequences of unknowns that can be matched and subsequently identified. The Fish Barcode of Life (FISH-BOL) campaign has the primary goal of gathering DNA barcode records for all the world''s fish species. As a contribution to FISH-BOL, we examined the degree to which DNA barcoding can discriminate marine fishes from the South China Sea.

Methodology/Principal Findings

DNA barcodes of cytochrome oxidase subunit I (COI) were characterized using 1336 specimens that belong to 242 species fishes from the South China Sea. All specimen provenance data (including digital specimen images and geospatial coordinates of collection localities) and collateral sequence information were assembled using Barcode of Life Data System (BOLD; www.barcodinglife.org). Small intraspecific and large interspecific differences create distinct genetic boundaries among most species. In addition, the efficiency of two mitochondrial genes, 16S rRNA (16S) and cytochrome b (cytb), and one nuclear ribosomal gene, 18S rRNA (18S), was also evaluated for a few select groups of species.

Conclusions/Significance

The present study provides evidence for the effectiveness of DNA barcoding as a tool for monitoring marine biodiversity. Open access data of fishes from the South China Sea can benefit relative applications in ecology and taxonomy.     

Systematic and evolutionary insights derived from mtDNA COI barcode diversity in the Decapoda (Crustacea: Malacostraca)

Background

Decapods are the most recognizable of all crustaceans and comprise a dominant group of benthic invertebrates of the continental shelf and slope, including many species of economic importance. Of the 17635 morphologically described Decapoda species, only 5.4% are represented by COI barcode region sequences. It therefore remains a challenge to compile regional databases that identify and analyse the extent and patterns of decapod diversity throughout the world.

Methodology/Principal Findings

We contributed 101 decapod species from the North East Atlantic, the Gulf of Cadiz and the Mediterranean Sea, of which 81 species represent novel COI records. Within the newly-generated dataset, 3.6% of the species barcodes conflicted with the assigned morphological taxonomic identification, highlighting both the apparent taxonomic ambiguity among certain groups, and the need for an accelerated and independent taxonomic approach. Using the combined COI barcode projects from the Barcode of Life Database, we provide the most comprehensive COI data set so far examined for the Order (1572 sequences of 528 species, 213 genera, and 67 families). Patterns within families show a general predicted molecular hierarchy, but the scale of divergence at each taxonomic level appears to vary extensively between families. The range values of mean K2P distance observed were: within species 0.285% to 1.375%, within genus 6.376% to 20.924% and within family 11.392% to 25.617%. Nucleotide composition varied greatly across decapods, ranging from 30.8 % to 49.4 % GC content.

Conclusions/Significance

Decapod biological diversity was quantified by identifying putative cryptic species allowing a rapid assessment of taxon diversity in groups that have until now received limited morphological and systematic examination. We highlight taxonomic groups or species with unusual nucleotide composition or evolutionary rates. Such data are relevant to strategies for conservation of existing decapod biodiversity, as well as elucidating the mechanisms and constraints shaping the patterns observed.     

Validation of the ITS2 Region as a Novel DNA Barcode for Identifying Medicinal Plant Species

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.     

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

Background

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.

Results

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.

Conclusion

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.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-264) contains supplementary material, which is available to authorized users.     

Conflicts of Interest among Authors of Clinical Practice Guidelines for Glycemic Control in Type 2 Diabetes Mellitus

Background

Conflict of interest (COI) is an important potential source of bias in the development of clinical practice guidelines (CPGs).

Objectives

To examine rates of disclosure of COI, including financial interests in companies that manufacture drugs that are recommended in CPGs on glycemic control in type 2 diabetes mellitus, and to explore the relationship between recommendations for specific drugs in a guideline and author COI.

Methods

We identified a cohort of relevant guidelines from the National Guideline Clearinghouse (NGC) and abstracted COI disclosures from all guideline authors for this observational, cross-sectional study. We determined which hypoglycemic drugs were recommended in each guideline, and explored the relationship between specific disclosures and whether a drug was recommended.

Results

Among 13 included guidelines, the percentage of authors with one or more financial disclosures varied from 0 to 94% (mean 44.2%), and was particularly high for two US-based guidelines (91% and 94%). Three guidelines disclosed no author financial COI. The percentage of authors with disclosures of financial interests in manufacturers of recommended drugs was also high (mean 30%). On average, 56% of manufacturers of patented drugs recommended in each guideline had one or more authors with a financial interest in their company. We did not find a significant relationship between financial interests and whether a drug was recommended in our sample; US-based guidelines were more likely to make recommendations for a specific drug compared to non-US based guidelines.

Discussion

Authors of this cohort of guidelines have financial interests directly related to the drugs that they are recommending. Although we did not find an association between author COI and drugs recommended in these guidelines and we cannot draw conclusions about the validity of the recommendations, the credibility of many of these guidelines is in doubt.     

Comparing the usefulness of distance, monophyly and character-based DNA barcoding methods in species identification: a case study of neogastropoda

Background

DNA barcoding has recently been proposed as a promising tool for the rapid species identification in a wide range of animal taxa. Two broad methods (distance and monophyly-based methods) have been used. One method is based on degree of DNA sequence variation within and between species while another method requires the recovery of species as discrete clades (monophyly) on a phylogenetic tree. Nevertheless, some issues complicate the use of both methods. A recently applied new technique, the character-based DNA barcode method, however, characterizes species through a unique combination of diagnostic characters.

Methodology/Principal Findings

Here we analyzed 108 COI and 102 16S rDNA sequences of 40 species of Neogastropoda from a wide phylogenetic range to assess the performance of distance, monophyly and character-based methods of DNA barcoding. The distance-based method for both COI and 16S rDNA genes performed poorly in terms of species identification. Obvious overlap between intraspecific and interspecific divergences for both genes was found. The “10× rule” threshold resulted in lumping about half of distinct species for both genes. The neighbour-joining phylogenetic tree of COI could distinguish all species studied. However, the 16S rDNA tree could not distinguish some closely related species. In contrast, the character-based barcode method for both genes successfully identified 100% of the neogastropod species included, and performed well in discriminating neogastropod genera.

Conclusions/Significance

This present study demonstrates the effectiveness of the character-based barcoding method for species identification in different taxonomic levels, especially for discriminating the closely related species. While distance and monophyly-based methods commonly use COI as the ideal gene for barcoding, the character-based approach can perform well for species identification using relatively conserved gene markers (e.g., 16S rDNA in this study). Nevertheless, distance and monophyly-based methods, especially the monophyly-based method, can still be used to flag species.     

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

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.