Optical barcoding of colloidal suspensions: applications in genomics, proteomics and drug discovery

The enormous amount of information generated through sequencing of the human genome has increased demands for more economical and flexible alternatives in genomics, proteomics and drug discovery. Many companies and institutions have recognised the potential of increasing the size and complexity of chemical libraries by producing large chemical libraries on colloidal support beads. Since colloid-based compounds in a suspension are randomly located, an encoding system such as optical barcoding is required to permit rapid elucidation of the compound structures. We describe in this article innovative methods for optical barcoding of colloids for use as support beads in both combinatorial and non-combinatorial libraries. We focus in particular on the difficult problem of barcoding extremely large libraries, which if solved, will transform the manner in which genomics, proteomics and drug discovery research is currently performed.

DNA-barcoding revela la existencia de un posible nuevo género del complejo Laurencia (Rodophyta, Ceramiales) en las islas Canarias

XIX Simposio de Botánica Criptogámica, Las Palmas de Gran Canaria, 24-28 de junio de 2013.

Barcoding human physical activity to assess chronic pain conditions.

BACKGROUND: Modern theories define chronic pain as a multidimensional experience - the result of complex interplay between physiological and psychological factors with significant impact on patients' physical, emotional and social functioning. The development of reliable assessment tools capable of capturing the multidimensional impact of chronic pain has challenged the medical community for decades. A number of validated tools are currently used in clinical practice however they all rely on self-reporting and are therefore inherently subjective. In this study we show that a comprehensive analysis of physical activity (PA) under real life conditions may capture behavioral aspects that may reflect physical and emotional functioning.¦METHODOLOGY: PA was monitored during five consecutive days in 60 chronic pain patients and 15 pain-free healthy subjects. To analyze the various aspects of pain-related activity behaviors we defined the concept of PA 'barcoding'. The main idea was to combine different features of PA (type, intensity, duration) to define various PA states. The temporal sequence of different states was visualized as a 'barcode' which indicated that significant information about daily activity can be contained in the amount and variety of PA states, and in the temporal structure of sequence. This information was quantified using complementary measures such as structural complexity metrics (information and sample entropy, Lempel-Ziv complexity), time spent in PA states, and two composite scores, which integrate all measures. The reliability of these measures to characterize chronic pain conditions was assessed by comparing groups of subjects with clinically different pain intensity.¦CONCLUSION: The defined measures of PA showed good discriminative features. The results suggest that significant information about pain-related functional limitations is captured by the structural complexity of PA barcodes, which decreases when the intensity of pain increases. We conclude that a comprehensive analysis of daily-life PA can provide an objective appraisal of the intensity of pain.

Heterobathmia pseuderiocrania Kristensen & Nielsen (Lepidoptera, Heterobathmiidae): identificación basada en DNA-barcoding y notas morfológicas e historia de vida de los estados inmaduros

Heterobathmia pseuderiocrania Kristensen & Nielsen (Lepidoptera, Heterobathmiidae): identification based on DNA-barcoding and notes on the morphology and life history of the immature stages. The larva morphology of the species Heterobathmia pseuderiocrania (Lepidoptera, Heterobathmiidae), a Nothofagus obliqua leafminer in Chile, is described. The tissue-feeding first and last instars are described. Also, the number of larval stages, some aspects of the biology and life cycle of the species are provided.

DNA barcoding of Corsican mayflies (Ephemeroptera) with implications on biogeography, systematics and biodiversity

Mayflies (Ephemeroptera) are known to generally present a high degree of insular endemism: half of the 28 species known from Corsica and Sardinia are considered as endemic. We sequenced the DNA barcode (a fragment of the mitochondrial COI gene) of 349 specimens from 50 localities in Corsica, Sardinia, continental Europe and North Africa. We reconstructed gene trees of eight genera or species groups representing the main mayfly families. Alternative topologies were built to test if our reconstructions suggested a single or multiple Corsican/Sardinian colonization event(s) in each genus or species group. A molecular clock calibrated with different evolution rates was used to try to link speciation processes with geological events. Our results confirm the high degree of endemism of Corsican and Sardinian mayflies and the close relationship between these two faunas. Moreover, we have evidence that the mayfly diversity of the two islands is highly underestimated as at least six new putative species occur on the two islands. We demonstrated that the Corsican and Sardinian mayfly fauna reveals a complex history mainly related to geological events. The Messinian Salinity Crisis, which is thought to have reduced marine barriers, thus facilitating gene flow between insular and continental populations, was detected as the most important event in the speciation of most lineages. Vicariance processes related to the split and rotation of the Corso-Sardinian microplate had a minor impact as they involved only two genera with limited dispersal and ecological range. Colonization events posterior to the Messinian Salinity Crisis had only marginal effects as we had indication of recent gene flow only in two clades. With very limited recent gene flow and a high degree of endemism, mayflies from Corsica and Sardinia present all the criteria for conservation prioritization.

Selection of candidate coding DNA barcoding regions for use on land plants

An in silico screen of 41 of the 81 coding regions of the Nicotiana plastid genome generated a shortlist of 12 candidates as DNA barcoding loci for land plants. These loci were evaluated for amplification and sequence variation against a reference set of 98 land plant taxa. The deployment of multiple primers and a modified multiplexed tandem polymerase chain reaction yielded 85–94% amplification across taxa, and mean sequence differences between sister taxa of 6.1 from 156 bases of accD to 22 from 493 bases of matK. We conclude that loci should be combined for effective diagnosis, and recommend further investigation of the following six loci: matK, rpoB, rpoC1, ndhJ, ycf5 and accD.

DNA barcoding of a large genus, Aspalathus L. (Fabaceae)

The DNA barcode potential of three regions (the nuclear ribosomal ITS and the plastid psbA-trnH and trnT-trnL intergenic spacers) was investigated for the plant genus Aspalathus L. (Fabaceac: Crotalarieae). Aspalathus is a large genus (278 species) that revealed low levels of DNA variation in phylogenetic studies. In a 51-species dataset for the psbA-trnH and ITS regions, 45%, and 16% of sequences respectively were identical to the sequence of at least one other species, with two species undiscriminated even when the two regions were combined. In contrast, trnT-trnL, discriminated between all species in this dataset. In a larger ITS and trnT-trnL dataset. including a further 82 species. 7 species in five pairwise comparisons remained Undiscriminated when the two regions were combined. Four of the five pairs of species not discriminated by sequence data were readily distinguished using a combination of qualitative and quantitative morphological data. The difficulty of barcoding in this group is increased by the presence of intraspecific variation in all three regions studied. In the case of psbA-trnH, three intraspecific samples had a sequence identical to at least one other species. Overall, psbA-trnH. currently a candidate for plant barcoding, was the least discriminatory region in our study.

DNA barcoding of Cyclamen species: another approach to species identification

Cyclamen is a diverse genus with some well defined and some intransigent species complexes. Here we report on an attempt to use DNA barcoding techniques to help evaluate species boundaries. DNA barcoding uses multiple samples, each from a different individual of a species, to generate a series of DNA sequences that can be compared for similarity.

Identification and lineage genotyping of South American trypanosomes using fluorescent fragment length barcoding

Trypanosoma cruzi and Trypanosoma rangeli are human-infective blood parasites, largely restricted to Central and South America. They also infect a wide range of wild and domestic mammals and are transmitted by a numerous species of triatomine bugs. There are significant overlaps in the host and geographical ranges of both species. The two species consist of a number of distinct phylogenetic lineages. A range of PCR-based techniques have been developed to differentiate between these species and to assign their isolates into lineages. However, the existence of at least six and five lineages within T. cruzi and T. rangeli, respectively, makes identification of the full range of isolates difficult and time consuming. Here we have applied fluorescent fragment length barcoding (FFLB) to the problem of identifying and genotyping T. cruzi, T. rangeli and other South American trypanosomes. This technique discriminates species on the basis of length polymorphism of regions of the rDNA locus. FFLB was able to differentiate many trypanosome species known from South American mammals: T. cruzi cruzi. T. cruzi marinkellei, T. dionisii-like, T. evansi, T. lewisi, T. rangeli, T. theileri and T. vivax. Furthermore, all five T. rangeli lineages and many T. cruzi lineages could be identified, except the hybrid lineages TcV and TcVI that could not be distinguished from lineages III and II respectively. This method also allowed identification of mixed infections of T. cruzi and T. rangeli lineages in naturally infected triatomine bugs. The ability of FFLB to genotype multiple lineages of T. cruzi and T. rangeli together with other trypanosome species, using the same primer sets is an advantage over other currently available techniques. Overall, these results demonstrate that FFLB is a useful method for species diagnosis, genotyping and understanding the epidemiology of American trypanosomes. (C) 2010 Elsevier B.V. All rights reserved.

DNA barcoding reveals hidden diversity in the Neotropical freshwater fish Piabina argentea (Characiformes: Characidae) from the Upper Parana Basin of Brazil

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

DNA barcoding of Podonomus (Chironomidae, Podonominae) enables stage association of a named species and reveals hidden diversity in Brazilian inselbergs

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Contrasting intra versus interspecies DNA sequence variation for representatives of the Batrachospermales (Rhodophyta): Insights from a DNA barcoding approach

Sixty-five accessions of the species-rich freshwater red algal order Batrachospermales were characterized through DNA sequencing of two regions: the mitochondrial cox1 gene (664 bp), which is proposed as the DNA barcode for red algae, and the UPA (universal plastid amplicon) marker (370 bp), which has been recently identified as a universally amplifying region of the plastid genome. upgma phenograms of both markers were consistent in their species-level relationships, although levels of sequence divergence were very different. Intraspecific variation of morphologically identified accessions for the cox1 gene ranged from 0 to 67 bp (divergences were highest for the two taxa with the greatest number of accessions; Batrachospermum helminthosum and Batrachospermum macrosporum); while in contrast, the more conserved universal plastid amplicon exhibited much lower intraspecific variation (generally 0-3 bp). Comparisons to previously published mitochondrial cox2-3 spacer sequences for B. helminthosum indicated that the cox1 gene and cox2-3 spacer were characterized by similar levels of sequence divergence, and phylogeographic patterns based on these two markers were consistent. The two taxa represented by the largest numbers of specimens (B. helminthosum and B. macrosporum) have cox1 intraspecific divergence values that are substantially higher than previously reported, but no morphological differences can be discerned at this time among the intraspecific groups revealed in the analyses. DNA barcode data, which are based on a short fragment of an organellar genome, need to be interpreted in conjunction with other taxonomic characters, and additional batrachospermalean taxa need to be analyzed in detail to be able to draw generalities regarding intraspecific variation in this order. Nevertheless, these analyses reveal a number of batrachospermalean taxa worthy of more detailed DNA barcode study, and it is predicted that such research will have a substantial effect on the taxonomy of species within the Batrachospermales in the future.

Can DNA barcoding accurately discriminate megadiverse Neotropical freshwater fish fauna?

Background: The megadiverse Neotropical freshwater ichthyofauna is the richest in the world with approximately 6,000 recognized species. Interestingly, they are distributed among only 17 orders, and almost 80% of them belong to only three orders: Characiformes, Siluriformes and Perciformes. Moreover, evidence based on molecular data has shown that most of the diversification of the Neotropical ichthyofauna occurred recently. These characteristics make the taxonomy and identification of this fauna a great challenge, even when using molecular approaches. In this context, the present study aimed to test the effectiveness of the barcoding methodology (COI gene) to identify the mega diverse freshwater fish fauna from the Neotropical region. For this purpose, 254 species of fishes were analyzed from the Upper Parana River basin, an area representative of the larger Neotropical region.Results: Of the 254 species analyzed, 252 were correctly identified by their barcode sequences (99.2%). The main K2P intra- and inter-specific genetic divergence values (0.3% and 6.8%, respectively) were relatively low compared with similar values reported in the literature, reflecting the higher number of closely related species belonging to a few higher taxa and their recent radiation. Moreover, for 84 pairs of species that showed low levels of genetic divergence (<2%), application of a complementary character-based nucleotide diagnostic approach proved useful in discriminating them. Additionally, 14 species displayed high intra-specific genetic divergence (>2%), pointing to at least 23 strong candidates for new species.Conclusions: Our study is the first to examine a large number of freshwater fish species from the Neotropical area, including a large number of closely related species. The results confirmed the efficacy of the barcoding methodology to identify a recently radiated, megadiverse fauna, discriminating 99.2% of the analyzed species. The power of the barcode sequences to identify species, even with low interspecific divergence, gives us an idea of the distribution of inter-specific genetic divergence in these megadiverse fauna. The results also revealed hidden genetic divergences suggestive of reproductive isolation and putative cryptic speciation in some species (23 candidates for new species). Finally, our study constituted an important contribution to the international Barcoding of Life (iBOL.org) project, providing barcode sequences for use in identification of these species by experts and non-experts, and allowing them to be available for use in other applications. © 2013 Pereira et al.; licensee BioMed Central Ltd.