Morphology and Phase Compositions of FePt and CoPt Nanoparticles Enriched with Noble Metal.

The article reveals for the first time the features of nanoparticle morphology, phase compositions, and their changes when heating FePt and CoPt nanoalloys. Nanoparticles were obtained by co-reduction of precursor solution mixtures with hydrazine hydrate. The features were found by a complex of methods of X-ray diffraction (in situ XRD and X-ray scattering), TEM HR, and cyclic voltammetry. In addition, adsorbometry results were obtained, and the stability of different nanocluster structures was calculated by the molecular dynamics method. There were only FCC solid solutions in the X-ray patterns of the FePt and CoPt nanoalloys. According to XRD, in the case of nanoparticle synthesis with Fe and Co content less than 10 at. %, the composition of solid solutions was close to or practically equal to the composition of the as-synthesized nanoparticles quantified by inductively coupled plasma optical emission spectrometry. For systems synthesis with Fe and Co content greater than the above, the solubility limits (SLs) of Fe and Co in Pt were set 11.4 ± 0.7 at. % and 17.5 ± 0.6 at. %, respectively. Therefore, there were non-registered XRD extra-phases (XRNDPh-1) in the systems when CFe,Co ≥ SL. This statement was supported by the results of TEM HR and X-ray scattering: the smallest nanocrystals (1-2 nm) and amorphous particles were found, which qualitatively agreed with the sorbometry and SAXS results. Molecular dynamics calculations of stability for FePt and CoPt alloys claimed the structures of the most stable phase corresponded to phase diagrams (A1 and L12). Specific peculiarities of the morphology and compositions of the solid solutions of nanoalloys were established: structural blockiness (domain) and composition heterogeneity, namely, platinum enrichment of internal (deep) layers and homogenization of the nanoalloy compositions at relatively low temperatures (130-200 °C). The suggested model of the formation of nanoalloys during the synthesis, qualitatively, was compliant with the results of electrochemical deposition of FePt films on the surface of various electrodes. When nanocrystals of solid solutions (C(Fe, Co) < SL) were heated above specific temperatures, there were phase transformations with the formation of two-phase regions, with solid solutions enriched with platinum or iron (non-registered XRD phase XRNDPh-2). The newly formed phase was most likely intermetallic compounds, FePt3, CoPt3. As a result of the study, the model was developed, taking into account the nanoscale of the particles: XRDPh (A1, FeaPt1-a) → XRDPh (A1, Fem×a-xPtm-m×a+x) + XRNDPh-2 (Fen×a+yPtn-n×a-y) (here, m + n = 1, m ≤ 1, n ≤ 1).

A reference library for Canadian invertebrates with 1.5 million barcodes, voucher specimens, and DNA samples.

The reliable taxonomic identification of organisms through DNA sequence data requires a well parameterized library of curated reference sequences. However, it is estimated that just 15% of described animal species are represented in public sequence repositories. To begin to address this deficiency, we provide DNA barcodes for 1,500,003 animal specimens collected from 23 terrestrial and aquatic ecozones at sites across Canada, a nation that comprises 7% of the planet's land surface. In total, 14 phyla, 43 classes, 163 orders, 1123 families, 6186 genera, and 64,264 Barcode Index Numbers (BINs; a proxy for species) are represented. Species-level taxonomy was available for 38% of the specimens, but higher proportions were assigned to a genus (69.5%) and a family (99.9%). Voucher specimens and DNA extracts are archived at the Centre for Biodiversity Genomics where they are available for further research. The corresponding sequence and taxonomic data can be accessed through the Barcode of Life Data System, GenBank, the Global Biodiversity Information Facility, and the Global Genome Biodiversity Network Data Portal.

Validation of COI metabarcoding primers for terrestrial arthropods.

Metabarcoding can rapidly determine the species composition of bulk samples and thus aids biodiversity and ecosystem assessment. However, it is essential to use primer sets that minimize amplification bias among taxa to maximize species recovery. Despite this fact, the performance of primer sets employed for metabarcoding terrestrial arthropods has not been sufficiently evaluated. This study tests the performance of 36 primer sets on a mock community containing 374 insect species. Amplification success was assessed with gradient PCRs and the 21 most promising primer sets selected for metabarcoding. These 21 primer sets were also tested by metabarcoding a Malaise trap sample. We identified eight primer sets, mainly those including inosine and/or high degeneracy, that recovered more than 95% of the species in the mock community. Results from the Malaise trap sample were congruent with the mock community, but primer sets generating short amplicons produced potential false positives. Taxon recovery from both mock community and Malaise trap sample metabarcoding were used to select four primer sets for additional evaluation at different annealing temperatures (40-60 °C) using the mock community. The effect of temperature varied by primer pair but overall it only had a minor effect on taxon recovery. This study reveals the weak performance of some primer sets employed in past studies. It also demonstrates that certain primer sets can recover most taxa in a diverse species assemblage. Thus, based our experimental set up, there is no need to employ several primer sets targeting the same gene region. We identify several suitable primer sets for arthropod metabarcoding, and specifically recommend BF3 + BR2, as it is not affected by primer slippage and provides maximal taxonomic resolution. The fwhF2 + fwhR2n primer set amplifies a shorter fragment and is therefore ideal when targeting degraded DNA (e.g., from gut contents).

Idiotypic and anti-idiotypic antibodies against polycyclic aromatic hydrocarbon in human blood serum are new biomarkers of lung cancer.

Evaluation of epidemiologic risk factor in relation to lung cancer invoked by polycyclic aromatic hydrocarbons has been inconsistent. To address this issue, we conducted a prospective evaluation of new biomarkers for lung cancer classified according levels of idiotypic and anti-idiotypic antibodies against polycyclic aromatic hydrocarbons in human blood serum. The blood serums of 557 lung cancer patients and 227 healthy donors were analysis of these antibodies by ELISA. Collected data were regrouped and analyzed by gender, smoking, and age as predictors of risk lung cancer factors. Also, the data of lung cancer patients were additionally analyzed by stages and types of lung cancer, surgery, and chemotherapy. It was suggested to use ratio of idiotypic and anti-idiotypic antibodies rather than distinguish level each of them separately. The ratio of levels in healthy people was 3.32 times higher than in lung cancer patients. This approach gave more precisely results and great prognostic value. The logistic regression model (AUC = 0.9) and neural networks (AUC = 0.95) were built to compare lung cancer patients and healthy donors by predictors. The ELISA data of 49 people random sampled from the originally ELISA data and ELISA data of 52 coal miners as a group of lung cancer risk were confirmed logistic regression model. So, suggested idiotypic and anti-idiotypic antibodies against polycyclic aromatic hydrocarbons were not only shown difference between healthy donors and lung cancer patients also elicited group of lung cancer risk among healthy people.

Using eDNA to biomonitor the fish community in a tropical oligotrophic lake.

Environmental DNA (eDNA) is an effective approach for detecting vertebrates and plants, especially in aquatic ecosystems, but prior studies have largely examined eDNA in cool temperate settings. By contrast, this study employs eDNA to survey the fish fauna in tropical Lake Bacalar (Mexico) with the additional goal of assessing the possible presence of invasive fishes, such as Amazon sailfin catfish and tilapia. Sediment and water samples were collected from eight stations in Lake Bacalar on three occasions over a 4-month interval. Each sample was stored in the presence or absence of lysis buffer to compare eDNA recovery. Short fragments (184-187 bp) of the cytochrome c oxidase I (COI) gene were amplified using fusion primers and then sequenced on Ion Torrent PGM or S5 before their source species were determined using a custom reference sequence database constructed on BOLD. In total, eDNA sequences were recovered from 75 species of vertebrates including 47 fishes, 15 birds, 7 mammals, 5 reptiles, and 1 amphibian. Although all species are known from this region, six fish species represent new records for the study area, while two require verification. Sequences for five species (2 birds, 2 mammals, 1 reptile) were only detected from sediments, while sequences from 52 species were only recovered from water. Because DNA from the Amazon sailfin catfish was not detected, we used a mock eDNA experiment to confirm our methods would enable its detection. In summary, we developed protocols that recovered eDNA from tropical oligotrophic aquatic ecosystems and confirmed their effectiveness in detecting fishes and diverse species of vertebrates.

Metabarcoding a diverse arthropod mock community.

Although DNA metabarcoding is an attractive approach for monitoring biodiversity, it is often difficult to detect all the species present in a bulk sample. In particular, sequence recovery for a given species depends on its biomass and mitome copy number as well as the primer set employed for PCR. To examine these variables, we constructed a mock community of terrestrial arthropods comprised of 374 species. We used this community to examine how species recovery was impacted when amplicon pools were constructed in four ways. The first two protocols involved the construction of bulk DNA extracts from different body segments (Bulk Abdomen, Bulk Leg). The other protocols involved the production of DNA extracts from single legs which were then merged prior to PCR (Composite Leg) or PCR-amplified separately (Single Leg) and then pooled. The amplicons generated by these four treatments were then sequenced on three platforms (Illumina MiSeq, Ion Torrent PGM and Ion Torrent S5). The choice of sequencing platform did not substantially influence species recovery, although the Miseq delivered the highest sequence quality. As expected, species recovery was most efficient from the Single Leg treatment because amplicon abundance varied little among taxa. Among the three treatments where PCR occurred after pooling, the Bulk Abdomen treatment produced a more uniform read abundance than the Bulk Leg or Composite Leg treatment. Primer choice also influenced species recovery and evenness. Our results reveal how variation in protocols can have substantial impacts on perceived diversity unless sequencing coverage is sufficient to reach an asymptote.

Approaches to integrating genetic data into ecological networks.

As molecular tools for assessing trophic interactions become common, research is increasingly focused on the construction of interaction networks. Here, we demonstrate three key methods for incorporating DNA data into network ecology and discuss analytical considerations using a model consisting of plants, insects, bats and their parasites from the Costa Rica dry forest. The simplest method involves the use of Sanger sequencing to acquire long sequences to validate or refine field identifications, for example of bats and their parasites, where one specimen yields one sequence and one identification. This method can be fully quantified and resolved and these data resemble traditional ecological networks. For more complex taxonomic identifications, we target multiple DNA loci, for example from a seed or fruit pulp sample in faeces. These networks are also well resolved but gene targets vary in resolution and quantification is difficult. Finally, for mixed templates such as faecal contents of insectivorous bats, we use DNA metabarcoding targeting two sequence lengths (157 and 407 bp) of one gene region and a MOTU, BLAST and BIN association approach to resolve nodes. This network type is complex to generate and analyse, and we discuss the implications of this type of resolution on network analysis. Using these data, we construct the first molecular-based network of networks containing 3,304 interactions between 762 nodes of eight trophic functions and involving parasitic, mutualistic and predatory interactions. We provide a comparison of the relative strengths and weaknesses of these data types in network ecology.

Expedited assessment of terrestrial arthropod diversity by coupling Malaise traps with DNA barcoding 1.

Monitoring changes in terrestrial arthropod communities over space and time requires a dramatic increase in the speed and accuracy of processing samples that cannot be achieved with morphological approaches. The combination of DNA barcoding and Malaise traps allows expedited, comprehensive inventories of species abundance whose cost will rapidly decline as high-throughput sequencing technologies advance. Aside from detailing protocols from specimen sorting to data release, this paper describes their use in a survey of arthropod diversity in a national park that examined 21 194 specimens representing 2255 species. These protocols can support arthropod monitoring programs at regional, national, and continental scales.

A Sequel to Sanger: amplicon sequencing that scales.

BACKGROUND: Although high-throughput sequencers (HTS) have largely displaced their Sanger counterparts, the short read lengths and high error rates of most platforms constrain their utility for amplicon sequencing. The present study tests the capacity of single molecule, real-time (SMRT) sequencing implemented on the SEQUEL platform to overcome these limitations, employing 658 bp amplicons of the mitochondrial cytochrome c oxidase I gene as a model system. RESULTS: By examining templates from more than 5000 species and 20,000 specimens, the performance of SMRT sequencing was tested with amplicons showing wide variation in GC composition and varied sequence attributes. SMRT and Sanger sequences were very similar, but SMRT sequencing provided more complete coverage, especially for amplicons with homopolymer tracts. Because it can characterize amplicon pools from 10,000 DNA extracts in a single run, the SEQUEL can reduce greatly reduce sequencing costs in comparison to first (Sanger) and second generation platforms (Illumina, Ion). CONCLUSIONS: SMRT analysis generates high-fidelity sequences from amplicons with varying GC content and is resilient to homopolymer tracts. Analytical costs are low, substantially less than those for first or second generation sequencers. When implemented on the SEQUEL platform, SMRT analysis enables massive amplicon characterization because each instrument can recover sequences from more than 5 million DNA extracts a year.

Correction: Authentication of Herbal Supplements Using Next-Generation Sequencing.

[This corrects the article DOI: 10.1371/journal.pone.0156426.].

Authentication of Herbal Supplements Using Next-Generation Sequencing.

BACKGROUND: DNA-based testing has been gaining acceptance as a tool for authentication of a wide range of food products; however, its applicability for testing of herbal supplements remains contentious. METHODS: We utilized Sanger and Next-Generation Sequencing (NGS) for taxonomic authentication of fifteen herbal supplements representing three different producers from five medicinal plants: Echinacea purpurea, Valeriana officinalis, Ginkgo biloba, Hypericum perforatum and Trigonella foenum-graecum. Experimental design included three modifications of DNA extraction, two lysate dilutions, Internal Amplification Control, and multiple negative controls to exclude background contamination. Ginkgo supplements were also analyzed using HPLC-MS for the presence of active medicinal components. RESULTS: All supplements yielded DNA from multiple species, rendering Sanger sequencing results for rbcL and ITS2 regions either uninterpretable or non-reproducible between the experimental replicates. Overall, DNA from the manufacturer-listed medicinal plants was successfully detected in seven out of eight dry herb form supplements; however, low or poor DNA recovery due to degradation was observed in most plant extracts (none detected by Sanger; three out of seven-by NGS). NGS also revealed a diverse community of fungi, known to be associated with live plant material and/or the fermentation process used in the production of plant extracts. HPLC-MS testing demonstrated that Ginkgo supplements with degraded DNA contained ten key medicinal components. CONCLUSION: Quality control of herbal supplements should utilize a synergetic approach targeting both DNA and bioactive components, especially for standardized extracts with degraded DNA. The NGS workflow developed in this study enables reliable detection of plant and fungal DNA and can be utilized by manufacturers for quality assurance of raw plant materials, contamination control during the production process, and the final product. Interpretation of results should involve an interdisciplinary approach taking into account the processes involved in production of herbal supplements, as well as biocomplexity of plant-plant and plant-fungal biological interactions.

Implications of hybridization, NUMTs, and overlooked diversity for DNA Barcoding of Eurasian ground squirrels.

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'.

Protocols for dry DNA storage and shipment at room temperature.

The globalization of DNA barcoding will require core analytical facilities to develop cost-effective, efficient protocols for the shipment and archival storage of DNA extracts and PCR products. We evaluated three dry-state DNA stabilization systems: commercial Biomatrica(®) DNAstable(®) plates, home-made trehalose and polyvinyl alcohol (PVA) plates on 96-well panels of insect DNA stored at 56 °C and at room temperature. Controls included unprotected samples that were stored dry at room temperature and at 56 °C, and diluted samples held at 4 °C and at -20 °C. PCR and selective sequencing were performed over a 4-year interval to test the condition of DNA extracts. Biomatrica(®) provided better protection of DNA at 56 °C and at room temperature than trehalose and PVA, especially for diluted samples. PVA was the second best protectant after Biomatrica(®) at room temperature, whereas trehalose was the second best protectant at 56 °C. In spite of lower PCR success, the DNA stored at -20 °C yielded longer sequence reads and stronger signal, indicating that temperature is a crucial factor for DNA quality which has to be considered especially for long-term storage. Although it is premature to advocate a transition to DNA storage at room temperature, dry storage provides an additional layer of security for frozen samples, protecting them from degradation in the event of freezer failure. All three forms of DNA preservation enable shipment of dry DNA and PCR products between barcoding facilities.

Potential use of DNA barcodes in regulatory science: identification of the U.S. Food and Drug Administration's "Dirty 22," contributors to the spread of foodborne pathogens.

The U.S. Food, Drug, and Cosmetic Act prohibits the distribution of food that is adulterated, and the regulatory mission of the U.S. Food and Drug Administration (FDA) is to enforce this Act. FDA field laboratories have identified the 22 most common pests that contribute to the spread of foodborne disease (the "Dirty 22"). The current method of detecting filth and extraneous material (tails, legs, carcasses, etc.) is visual inspection using microscopy. Because microscopy can be time-consuming and may yield inaccurate and/or nonspecific results due to lack of expertise, an alternative method of detecting these adulterants is needed. In this study, we sequenced DNA from the 5' region of the cytochrome oxidase I gene of these 22 common pests that contribute to the spread of foodborne pathogens. Here, we describe the generation of DNA barcodes for all 22 species. To date, this is the first attempt to develop a sequence-based regulatory database and systematic primer strategy to identify these FDA-targeted species. DNA barcoding can be a powerful tool that can aid the FDA in promoting the protection and safety of the U.S. food supply.

DNA barcoding in mammals.

DNA barcoding provides an operational framework for mammalian taxonomic identification and cryptic species discovery. Focused effort to build a reference library of genetic data has resulted in the assembly of over 35 K mammalian cytochrome c oxidase subunit I sequences and outlined the scope of mammal-related barcoding projects. Based on the above experience, this chapter recounts three typical methodological pathways involved in mammalian barcoding: routine methods aimed at assembling the reference sequence library from high quality samples, express approaches used to attain cheap and fast taxonomic identifications for applied purposes, and forensic techniques employed when dealing with degraded material. Most of the methods described are applicable to a range of vertebrate taxa outside Mammalia.

FISH-BOL and seafood identification: geographically dispersed case studies reveal systemic market substitution across Canada.

BACKGROUND AND AIMS: The Fish Barcode of Life campaign involves a broad international collaboration among scientists working to advance the identification of fishes using DNA barcodes. With over 25% of the world's known ichthyofauna currently profiled, forensic identification of seafood products is now feasible and is becoming routine. MATERIALS AND METHODS: Driven by growing consumer interest in the food supply, investigative reporters from five different media establishments procured seafood samples (n = 254) from numerous retail establishments located among five Canadian metropolitan areas between 2008 and 2010. The specimens were sent to the Canadian Centre for DNA Barcoding for analysis. By integrating the results from these individual case studies in a summary analysis, we provide a broad perspective on seafood substitution across Canada. RESULTS: Barcodes were recovered from 93% of the samples (n = 236), and identified using the Barcode of Life Data Systems "species identification" engine ( www.barcodinglife.org ). A 99% sequence similarity threshold was employed as a conservative matching criterion for specimen identification to the species level. Comparing these results against the Canadian Food Inspection Agency's "Fish List" a guideline to interpreting "false, misleading or deceptive" names (as per s 27 of the Fish Inspection regulations) demonstrated that 41% of the samples were mislabeled. Most samples were readily identified; however, this was not true in all cases because some samples had no close match. Others were ambiguous due to limited barcode resolution (or imperfect taxonomy) observed within a few closely related species complexes. The latter cases did not significantly impact the results because even the partial resolution achieved was sufficient to demonstrate mislabeling. CONCLUSION: This work highlights the functional utility of barcoding for the identification of diverse market samples. It also demonstrates how barcoding serves as a bridge linking scientific nomenclature with approved market names, potentially empowering regulatory bodies to enforce labeling standards. By synchronizing taxonomic effort with sequencing effort and database curation, barcoding provides a molecular identification resource of service to applied forensics.

A single-laboratory validated method for the generation of DNA barcodes for the identification of fish for regulatory compliance.

The U.S. Food and Drug Administration is responsible for ensuring that the nation's food supply is safe and accurately labeled. This task is particularly challenging in the case of seafood where a large variety of species are marketed, most of this commodity is imported, and processed product is difficult to identify using traditional morphological methods. Reliable species identification is critical for both foodborne illness investigations and for prevention of deceptive practices, such as those where species are intentionally mislabeled to circumvent import restrictions or for resale as species of higher value. New methods that allow accurate and rapid species identifications are needed, but any new methods to be used for regulatory compliance must be both standardized and adequately validated. "DNA barcoding" is a process by which species discriminations are achieved through the use of short, standardized gene fragments. For animals, a fragment (655 base pairs starting near the 5' end) of the cytochrome c oxidase subunit 1 mitochondrial gene has been shown to provide reliable species level discrimination in most cases. We provide here a protocol with single-laboratory validation for the generation of DNA barcodes suitable for the identification of seafood products, specifically fish, in a manner that is suitable for FDA regulatory use.

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.

Express barcodes: racing from specimen to identification.

Although devices combining microfluidic and advanced sequencing technologies promise a future where one can generate a DNA barcode in minutes, current analytical regimes typically involve workflows that extend over 2 days. Here we describe simple protocols enabling the advance from a specimen to barcode-based identification in less than 2 h. The protocols use frozen or lyophilized reagents that can be prepackaged into 'kits' and support barcode analysis across the animal kingdom. The analytical procedure allows 5 min for DNA extraction, 25 min for polymerase chain reaction amplification of the barcode region, 25 min for cycle-sequencing, 10 min for cleanup, 45 min for capillary sequencing and 5 min for trace file analysis to complete DNA-based identification. This study involved the comparison of varied DNA preservation and extraction methods, and evaluated Taq polymerases with high processivity and resistance to inhibitors.

Assembling DNA barcodes. Analytical protocols.

The Barcode of Life initiative represents an ambitious effort to develop an identification system for eukaryotic life based on the analysis of sequence diversity in short, standardized gene regions. Work is furthest advanced for members of the animal kingdom. In this case, a target gene region has been selected (cytochrome c oxidase I) and pilot studies have validated its effectiveness in species discovery and identification. Based on these positive results, there is now a growing effort to both gather barcode records on a large-scale for members of this kingdom and to identify target barcode regions for the other kingdoms of eukaryotes. In this chapter, we detail the protocols involved in the assembly of DNA barcode records for members of the animal kingdom, but many of these approaches are of more general application.