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


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          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.

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          A universal DNA mini-barcode for biodiversity analysis

          Background The goal of DNA barcoding is to develop a species-specific sequence library for all eukaryotes. A 650 bp fragment of the cytochrome c oxidase 1 (CO1) gene has been used successfully for species-level identification in several animal groups. It may be difficult in practice, however, to retrieve a 650 bp fragment from archival specimens, (because of DNA degradation) or from environmental samples (where universal primers are needed). Results We used a bioinformatics analysis using all CO1 barcode sequences from GenBank and calculated the probability of having species-specific barcodes for varied size fragments. This analysis established the potential of much smaller fragments, mini-barcodes, for identifying unknown specimens. We then developed a universal primer set for the amplification of mini-barcodes. We further successfully tested the utility of this primer set on a comprehensive set of taxa from all major eukaryotic groups as well as archival specimens. Conclusion In this study we address the important issue of minimum amount of sequence information required for identifying species in DNA barcoding. We establish a novel approach based on a much shorter barcode sequence and demonstrate its effectiveness in archival specimens. This approach will significantly broaden the application of DNA barcoding in biodiversity studies.
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            Critical factors for assembling a high volume of DNA barcodes.

            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.
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              Ultrasequencing of the meiofaunal biosphere: practice, pitfalls and promises.

              Biodiversity assessment is the key to understanding the relationship between biodiversity and ecosystem functioning, but there is a well-acknowledged biodiversity identification gap related to eukaryotic meiofaunal organisms. Meiofaunal identification is confounded by the small size of taxa, morphological convergence and intraspecific variation. However, the most important restricting factor in meiofaunal ecological research is the mismatch between diversity and the number of taxonomists that are able to simultaneously identify and catalogue meiofaunal diversity. Accordingly, a molecular operational taxonomic unit (MOTU)-based approach has been advocated for en mass meiofaunal biodiversity assessment, but it has been restricted by the lack of throughput afforded by chain termination sequencing. Contemporary pyrosequencing offers a solution to this problem in the form of environmental metagenetic analyses, but this represents a novel field of biodiversity assessment. Here, we provide an overview of meiofaunal metagenetic analyses, ranging from sample preservation and DNA extraction to PCR, sequencing and the bioinformatic interrogation of multiple, independent samples using 454 Roche sequencing platforms. We report two examples of environmental metagenetic nuclear small subunit 18S (nSSU) analyses of marine and tropical rainforest habitats and provide critical appraisals of the level of putative recombinant DNA molecules (chimeras) in metagenetic data sets. Following stringent quality control measures, environmental metagenetic analyses achieve MOTU formation across the eukaryote domain of life at a fraction of the time and cost of traditional approaches. The effectiveness of Roche 454 sequencing brings substantial advantages to studies aiming to elucidate the molecular genetic richness of not only meiofaunal, but also all complex eukaryotic communities.

                Author and article information

                Role: Editor
                PLoS One
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                13 April 2011
                : 6
                : 4
                [1 ]Biodiversity Institute of Ontario, Department of Integrative Biology, University of Guelph, Guelph, Ontario, Canada
                [2 ]Environment Canada, Canadian Rivers Institute, Department of Biology, University of New Brunswick, Fredericton, New Brunswick, Canada
                King Abdullah University of Science and Technology, Saudi Arabia
                Author notes

                Conceived and designed the experiments: MH SS XZ GACS DJB. Performed the experiments: MH SS XZ GACS. Analyzed the data: MH GACS. Contributed reagents/materials/analysis tools: MH DJB. Wrote the paper: MH. Edited the manuscript: SS DJB.

                Hajibabaei et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                Page count
                Pages: 7
                Research Article
                Environmental Protection



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