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      Indigenous species barcode database improves the identification of zooplankton

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          Abstract

          Incompleteness and inaccuracy of DNA barcode databases is considered an important hindrance to the use of metabarcoding in biodiversity analysis of zooplankton at the species-level. Species barcoding by Sanger sequencing is inefficient for organisms with small body sizes, such as zooplankton. Here mitochondrial cytochrome c oxidase I ( COI) fragment barcodes from 910 freshwater zooplankton specimens (87 morphospecies) were recovered by a high-throughput sequencing platform, Ion Torrent PGM. Intraspecific divergence of most zooplanktons was < 5%, except Branchionus leydign (Rotifer, 14.3%), Trichocerca elongate (Rotifer, 11.5%), Lecane bulla (Rotifer, 15.9%), Synchaeta oblonga (Rotifer, 5.95%) and Schmackeria forbesi (Copepod, 6.5%). Metabarcoding data of 28 environmental samples from Lake Tai were annotated by both an indigenous database and NCBI Genbank database. The indigenous database improved the taxonomic assignment of metabarcoding of zooplankton. Most zooplankton (81%) with barcode sequences in the indigenous database were identified by metabarcoding monitoring. Furthermore, the frequency and distribution of zooplankton were also consistent between metabarcoding and morphology identification. Overall, the indigenous database improved the taxonomic assignment of zooplankton.

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          A new versatile primer set targeting a short fragment of the mitochondrial COI region for metabarcoding metazoan diversity: application for characterizing coral reef fish gut contents

          Introduction The PCR-based analysis of homologous genes has become one of the most powerful approaches for species detection and identification, particularly with the recent availability of Next Generation Sequencing platforms (NGS) making it possible to identify species composition from a broad range of environmental samples. Identifying species from these samples relies on the ability to match sequences with reference barcodes for taxonomic identification. Unfortunately, most studies of environmental samples have targeted ribosomal markers, despite the fact that the mitochondrial Cytochrome c Oxidase subunit I gene (COI) is by far the most widely available sequence region in public reference libraries. This is largely because the available versatile (“universal”) COI primers target the 658 barcoding region, whose size is considered too large for many NGS applications. Moreover, traditional barcoding primers are known to be poorly conserved across some taxonomic groups. Results We first design a new PCR primer within the highly variable mitochondrial COI region, the “mlCOIintF” primer. We then show that this newly designed forward primer combined with the “jgHCO2198” reverse primer to target a 313 bp fragment performs well across metazoan diversity, with higher success rates than versatile primer sets traditionally used for DNA barcoding (i.e. LCO1490/HCO2198). Finally, we demonstrate how the shorter COI fragment coupled with an efficient bioinformatics pipeline can be used to characterize species diversity from environmental samples by pyrosequencing. We examine the gut contents of three species of planktivorous and benthivorous coral reef fish (family: Apogonidae and Holocentridae). After the removal of dubious COI sequences, we obtained a total of 334 prey Operational Taxonomic Units (OTUs) belonging to 14 phyla from 16 fish guts. Of these, 52.5% matched a reference barcode (>98% sequence similarity) and an additional 32% could be assigned to a higher taxonomic level using Bayesian assignment. Conclusions The molecular analysis of gut contents targeting the 313 COI fragment using the newly designed mlCOIintF primer in combination with the jgHCO2198 primer offers enormous promise for metazoan metabarcoding studies. We believe that this primer set will be a valuable asset for a range of applications from large-scale biodiversity assessments to food web studies.
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            Bias in template-to-product ratios in multitemplate PCR.

            Bias introduced by the simultaneous amplification of specific genes from complex mixtures of templates remains poorly understood. To explore potential causes and the extent of bias in PCR amplification of 16S ribosomal DNAs (rDNAs), genomic DNAs of two closely and one distantly related bacterial species were mixed and amplified with universal, degenerate primers. Quantification and comparison of template and product ratios showed that there was considerable and reproducible overamplification of specific templates. Variability between replicates also contributed to the observed bias but in a comparatively minor way. Based on these initial observations, template dosage and differences in binding energies of permutations of the degenerate, universal primers were tested as two likely causes of this template-specific bias by using 16S rDNA templates modified by site-directed mutagenesis. When mixtures of mutagenized templates containing AT- and GC-rich priming sites were used, templates containing the GC-rich permutation amplified with higher efficiency, indicating that different primer binding energies may to a large extent be responsible for overamplification. In contrast, gene copy number was found to be an unlikely cause of the observed bias. Similarly, amplification from DNA extracted from a natural community to which different amounts of genomic DNA of a single bacterial species were added did not affect relative product ratios. Bias was reduced considerably by using high template concentrations, by performing fewer cycles, and by mixing replicate reaction preparations.
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              Environmental Barcoding: A Next-Generation Sequencing Approach for Biomonitoring Applications Using River Benthos

              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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                Author and article information

                Contributors
                Role: Data curationRole: Project administrationRole: SoftwareRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: SupervisionRole: Writing – review & editing
                Role: Formal analysisRole: Methodology
                Role: InvestigationRole: Methodology
                Role: Data curationRole: Investigation
                Role: ValidationRole: Writing – review & editing
                Role: ConceptualizationRole: ValidationRole: Writing – original draft
                Role: ResourcesRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                4 October 2017
                2017
                : 12
                : 10
                : e0185697
                Affiliations
                [1 ] State Key Laboratory of Pollution Control & Resource Reuse, School of the Environment, Nanjing University, Nanjing, P. R. China
                [2 ] Nanjing Institute of Environmental Sciences, Ministry of Environmental Protection, Nanjing, China
                [3 ] School for Environment and Sustainability, University of Michigan, Ann Arbor, MI, United States of America
                National Taiwan Ocean University, TAIWAN
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0001-8974-9963
                Article
                PONE-D-17-01931
                10.1371/journal.pone.0185697
                5627919
                28977035
                69ea667d-9c12-4715-88dd-c58ff2d9dff9
                © 2017 Yang 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.

                History
                : 15 January 2017
                : 18 September 2017
                Page count
                Figures: 6, Tables: 0, Pages: 15
                Funding
                Funded by: Jiangsu Province Funds for Distinguished Young Scientists
                Award ID: BK20130015
                Award Recipient :
                Funded by: Major Science and Technology Program for Water Pollution Control and Treatment
                Award ID: Grant# 2012ZX07506-003
                Award Recipient :
                Funded by: Major Science and Technology Program for Water Pollution Control and Treatment
                Award ID: 2012ZX07101-007-01
                Award Recipient :
                For support, we thank Science Foundation of Jiangsu Province (BK20130015) and China Major Science and Technology Program for Water Pollution Control and Treatment (Grant #2012ZX07506-003 and 2012ZX07101-007). X.Z. was supported by the Fundamental Research Funds for the Central Universities. The research was also supported by the Collaborative Innovation Center for Regional Environmental Quality. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Research and Analysis Methods
                Database and Informatics Methods
                Biological Databases
                Sequence Databases
                Research and Analysis Methods
                Database and Informatics Methods
                Bioinformatics
                Sequence Analysis
                Sequence Databases
                Biology and Life Sciences
                Organisms
                Eukaryota
                Animals
                Invertebrates
                Plankton
                Zooplankton
                Biology and life sciences
                Molecular biology
                Molecular biology techniques
                Sequencing techniques
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                Sequencing techniques
                DNA sequencing
                Biology and Life Sciences
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                Molecular Biology
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