Metabarcoding and Metagenomics最新文献

{"title":"Ciliate SSU-rDNA reference alignments and trees for phylogenetic placements of metabarcoding data","authors":"Ľ. Rajter, M. Dunthorn","doi":"10.3897/mbmg.5.69602","DOIUrl":"https://doi.org/10.3897/mbmg.5.69602","url":null,"abstract":"Although ciliates are one of the most dominant microbial eukaryotic groups in many environments, there is a lack of updated global ciliate alignments and reference trees that can be used for phylogenetic placement methods to analyze environmental metabarcoding data. Here we fill this gap by providing reference alignments and trees for those ciliates taxa with available SSU-rDNA sequences derived from identified species. Each alignment contains 478 ciliate and six outgroup taxa, and they were made using different masking strategies for alignment positions (unmasked, masked and masked except the hypervariable V4 region). We constrained the monophyly of the major ciliate groups based on the recently updated classification of protists and based on phylogenomic data. Taxa of uncertain phylogenetic position were kept unconstrained, except for Mesodinium species that we constrained to form a clade with the Litostomatea. These ciliate reference alignments and trees can be used to perform taxonomic assignments of metabarcoding data, discover novel ciliate clades, estimate species richness, and overlay measured ecological parameters onto the phylogenetic placements.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70411952","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"How can integrated morphotaxonomy- and metabarcoding-based diatom assemblage analyses best contribute to the ecological assessment of streams?","authors":"Panayiota Pissaridou, M. Cantonati, A. Bouchez, I. Tziortzis, G. Dörflinger, M. Vasquez","doi":"10.3897/mbmg.5.68438","DOIUrl":"https://doi.org/10.3897/mbmg.5.68438","url":null,"abstract":"Environmental conditions, such as nutrient concentrations, salinity, elevation etc., shape diatom assemblages of periphytic biofilms. These assemblages respond rapidly to environmental changes, a fact which makes diatoms valuable bioindicators. Hence, freshwater biomonitoring programmes currently use diatom indices (e.g. EU Water Framework Directive - WFD). To date, microscopy-based assessments require high taxonomic expertise for diatom identification at the species level. High-throughput technologies now provide cost-effective identification approaches that are promising, complementary or alternative tools for bioassessment. The suitability of the metabarcoding method is evaluated for the first time in the Cyprus streams WFD monitoring network, an eastern Mediterranean country with many endemic species and results are compared to the results acquired from the morphotaxonomic analysis. Morphotaxonomic identification was conducted microscopically, using the most updated taxonomic concepts, literature and online resources. At the same time, DNA metabarcoding involved the use of the rbcL 312 bp barcode, high-throughput sequencing and bioinformatic analysis. The ecological status was calculated using the IPS Index. Results show a positive correlation between morpho-taxonomic and molecular IPS scores. Discrepancies between the two methodologies are related to the limitations of both techniques. This study confirmed that Fistulifera saprophila can have a crucial role in key differences observed, as it negatively influences IPS scores and microscopy methods frequently overlook it. Importantly, gaps in the DNA barcoding reference databases lead to a positive overestimation in IPS scores. Overall, we conclude that DNA metabarcoding offsets the morphotaxonomic methodology for the ecological quality assessment of freshwaters.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70411895","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Corrigendum: Using DNA metabarcoding for assessing chironomid diversity and community change in mosquito controlled temporary wetlands. MBMG 2: e21060. https://doi.org/10.3897/mbmg.2.21060","authors":"K. Theissinger, Anna Kästel, Vasco Elbrecht, Jenny Makkonen, S. Michiels, S. Schmidt, Stefanie Allgeier, F. Leese, C. Brühl","doi":"10.3897/mbmg.4.60854","DOIUrl":"https://doi.org/10.3897/mbmg.4.60854","url":null,"abstract":"Chironomids have been proposed as important indicators for monitoring freshwater ecosystems, however, morphological determination is very challenging. In this study, we investigated the effectiveness of metabarcoding for chironomid diversity assessment and tested the retrieved chironomid operational taxonomic units (OTUs) for possible changes in relative abundance and species diversity in relation to mosquito control actions in four temporary wetlands. The biocide Bacillus thuringiensis var. israelensis (Bti) is widely applied for mosquito control in temporary wetlands of the German Upper Rhine Valley. Even though Bti is considered environmentally friendly, several studies have shown non-target effects on chironomids, a key food resource in wetland ecosystems Three of the studied wetlands were, for the first year after 20 years of Bti treatment, partly left Bti-untreated in a split field design, and one wetland has never been treated with Bti. Our metabarcoding approach detected 54 chironomid OTUs across all study sites, of which 70% could be identified to species level comparisons against the BOLD database. We showed that metabarcoding increased chironomid species determination by 70%. However, we found only minor significant effects of Bti on the chironomid community composition. Subsequent studies will have to address if and how the chironomid community composition might change in the now Bti-untreated temporary wetlands to assess effects of Bti.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46669511","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Alignment-free classification of COI DNA barcode data with the Python package Alfie","authors":"C. Nugent, S. Adamowicz","doi":"10.3897/mbmg.4.55815","DOIUrl":"https://doi.org/10.3897/mbmg.4.55815","url":null,"abstract":"Characterization of biodiversity from environmental DNA samples and bulk metabarcoding data is hampered by off-target sequences that can confound conclusions about a taxonomic group of interest. Existing methods for isolation of target sequences rely on alignment to existing reference barcodes, but this can bias results against novel genetic variants. Effectively parsing targeted DNA barcode data from off-target noise improves the quality of biodiversity estimates and biological conclusions by limiting subsequent analyses to a relevant subset of available data. Here, we present Alfie, a Python package for the alignment-free classification of cytochrome c oxidase subunit I (COI) DNA barcode sequences to taxonomic kingdoms. The package determines k-mer frequencies of DNA sequences, and the frequencies serve as input for a neural network classifier that was trained and tested using ~58,000 publicly available COI sequences. The classifier was designed and optimized through a series of tests that allowed for the optimal set of DNA k-mer features and optimal machine learning algorithm to be selected. The neural network classifier rapidly assigns COI sequences of varying lengths to kingdoms with greater than 99% accuracy and is shown to generalize effectively and make accurate predictions about data from previously unseen taxonomic classes. The package contains an application programming interface that allows the Alfie package’s functionality to be extended to different DNA sequence classification tasks to suit a user’s need, including classification of different genes and barcodes, and classification to different taxonomic levels. Alfie is free and publicly available through GitHub (https://github.com/CNuge/alfie) and the Python package index (https://pypi.org/project/alfie/).","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":"24 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91395760","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Spatial structure of fungal DNA assemblages revealed with eDNA metabarcoding in a forest river network in western Japan","authors":"Shunsuke Matsuoka, Yoriko Sugiyama, Hirotoshi Sato, Izumi Katano, Ken Harada, H. Doi","doi":"10.3897/mbmg.3.36335","DOIUrl":"https://doi.org/10.3897/mbmg.3.36335","url":null,"abstract":"Growing evidence has revealed high diversity and spatial heterogeneity of fungal communities in local habitats of terrestrial ecosystems. Recently, the analysis of environmental DNA has been undertaken to study the biodiversity of organisms, such as animals and plants, in both aquatic and terrestrial habitats. In the present study, we investigated fungal DNA assemblages and their spatial structure using environmental DNA metabarcoding targeting the internal transcribed spacer 1 (ITS1) region of the rRNA gene cluster in habitats across different branches of rivers in forest landscapes. A total of 1,956 operational taxonomic units (OTUs) were detected. Of these, 770 were assigned as Ascomycota, 177 as Basidiomycota, and 38 as Chytridiomycota. The river water was found to contain functionally diverse OTUs of both aquatic and terrestrial fungi, such as plant decomposers and mycorrhizal fungi. These fungal DNA assemblages were more similar within, rather than between, river branches. In addition, the assemblages were more similar between spatially closer branches. This spatial structuring was significantly associated with geographic distances but not with vegetation of the catchment area and the elevation at the sampling points. Our results imply that information on the terrestrial and aquatic fungal compositions of watersheds, and therefore their spatial structure, can be obtained by investigating the fungal DNA assemblages in river water.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41462589","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Advancing the use of molecular methods for routine freshwater macroinvertebrate biomonitoring – the need for calibration experiments","authors":"Rosetta C. Blackman, E. Mächler, F. Altermatt, A. Arnold, P. Beja, P. Boets, Bastian Egeter, Vasco Elbrecht, A. Filipe, J. Jones, Jan-Niklas Macher, M. Majaneva, F. Martins, Cesc Múrria, Kristian Meissner, J. Pawłowski, Paul Schmidt Yáñez, V. Zizka, F. Leese, B. Price, Kristy Deiner","doi":"10.3897/MBMG.3.34735","DOIUrl":"https://doi.org/10.3897/MBMG.3.34735","url":null,"abstract":"Over the last decade, steady advancements have been made in the use of DNA-based methods for detection of species in a wide range of ecosystems. This progress has culminated in molecular monitoring methods being employed for the detection of several species for enforceable management purposes of endangered, invasive, and illegally harvested species worldwide. However, the routine application of DNA-based methods to monitor whole communities (typically a metabarcoding approach) in order to assess the status of ecosystems continues to be limited. In aquatic ecosystems, the limited use is particularly true for macroinvertebrate communities. As part of the DNAqua-Net consortium, a structured discussion was initiated with the aim to identify potential molecular methods for freshwater macroinvertebrate community assessment and identify important knowledge gaps for their routine application. We focus on three complementary DNA sources that can be metabarcoded: 1) DNA from homogenised samples (bulk DNA), 2) DNA extracted from sample preservative (fixative DNA), and 3) environmental DNA (eDNA) from water or sediment. We provide a brief overview of metabarcoding macroinvertebrate communities from each DNA source and identify challenges for their application to routine monitoring. To advance the utilisation of DNA-based monitoring for macroinvertebrates, we propose an experimental design template for a series of methodological calibration tests. The template compares sources of DNA with the goal of identifying the effects of molecular processing steps on precision and accuracy. Furthermore, the same samples will be morphologically analysed, which will enable the benchmarking of molecular to traditional processing approaches. In doing so we hope to highlight pathways for the development of DNA-based methods for the monitoring of freshwater macroinvertebrates.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48151912","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Molecular versus morphological data for benthic diatoms biomonitoring in Northern Europe freshwater and consequences for ecological status","authors":"Bonnie Bailet, A. Bouchez, A. Franc, J. Frigerio, F. Keck, S. Karjalainen, F. Rimet, S. Schneider, M. Kahlert","doi":"10.3897/MBMG.3.34002","DOIUrl":"https://doi.org/10.3897/MBMG.3.34002","url":null,"abstract":"Diatoms are known to be efficient bioindicators for water quality assessment because of their rapid response to environmental pressures and their omnipresence in water bodies. The identification of benthic diatoms communities in the biofilm, coupled with quality indices such as the Indice de polluosensibilité spécifique (IPS) can be used for biomonitoring purposes in freshwater. However, the morphological identification and counting of diatoms species under the microscope is time-consuming and requires extensive expertise to deal with a constantly evolving taxonomy. In response, a molecular-based and potentially more cost-effective method has been developed, coupling high-throughput sequencing and DNA metabarcoding. The method has already been tested for water quality assessment with diatoms in Central Europe. In this study, we applied both the traditional and molecular methods on 180 biofilms samples from Northern Europe (rivers and lakes of Fennoscandia and Iceland). The DNA metabarcoding data were obtained on two different DNA markers, the 18S-V4 and rbcL barcodes, with the NucleoSpin Soil kit for DNA extraction and sequenced on an Ion Torrent PGM platform. We assessed the ability of the molecular method to produce species inventories, IPS scores and ecological status class comparable to the ones generated by the traditional morphology-based approach. The two methods generated correlated but significantly different IPS scores and ecological status assessment. The observed deviations are explained by presence/absence and abundance discrepancies in the species inventories, mainly due to the incompleteness of the barcodes reference databases, primer bias and strictness of the bioinformatic pipeline. Abundance discrepancies are less common than presence/absence discrepancies but have a greater effect on the ecological assessment. Missing species in the reference databases are mostly acidophilic benthic diatoms species, typical of the low pH waters of Northern Europe. The two different DNA markers also generated significantly different ecological status assessments. The use of the 18S-V4 marker generates more species inventories discrepancies, but achieves an ecological assessment more similar to the traditional morphology-based method. Further development of the metabarcoding method is needed for its use in environmental assessment. For its application in Northern Europe, completion and curation of reference databases are necessary, as well as evaluation of the currently available bioinformatics pipelines. New indices, fitted for environmental biomonitoring, should also be developed directly from molecular data.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-06-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44082933","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Assessing pollution of aquatic environments with diatoms’ DNA metabarcoding: experience and developments from France water framework directive networks","authors":"V. Valentin, Rimet Frédéric, D. Isabelle, Monnier Olivier, R. Yorick, Bouchez Agnès","doi":"10.3897/mbmg.3.39646","DOIUrl":"https://doi.org/10.3897/mbmg.3.39646","url":null,"abstract":"Ecological status assessment of watercourses is based on the calculation of quality indices using pollution sensitivity of targeted biological groups, including diatoms. The determination and quantification of diatom species is generally based on microscopic morphological identification, which requires expertise and is time-consuming and costly. In Europe, this morphological approach is legally imposed by standards and regulatory decrees by the Water Framework Directive (WFD). Over the past decade, a DNA-based molecular biology approach has newly been developed to identify species based on genetic criteria rather than morphological ones (i.e. DNA metabarcoding). In combination with high throughput sequencing technologies, metabarcoding makes it possible both to identify all species present in an environmental sample and to process several hundred samples in parallel. This article presents the results of two recent studies carried out on the WFD networks of rivers of Mayotte (2013–2018) and metropolitan France (2016–2018). These studies aimed at testing the potential application of metabarcoding for biomonitoring in the context of the WFD. We discuss the various methodological developments and optimisations that have been made to make the taxonomic inventories of diatoms produced by metabarcoding more reliable, particularly in terms of species quantification. We present the results of the application of this DNA approach on more than 500 river sites, comparing them with those obtained using the standardised morphological method. Finally, we discuss the potential of metabarcoding for routine application, its limits of application and propose some recommendations for future implementation in WFD.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42129805","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Metabarcoding of soil nematodes: the importance of taxonomic coverage and availability of reference sequences in choosing suitable marker(s)","authors":"Mohammed Ahmed, M. Back, T. Prior, G. Karssen, R. Lawson, Ian Adams, M. Sapp","doi":"10.3897/mbmg.3.36408","DOIUrl":"https://doi.org/10.3897/mbmg.3.36408","url":null,"abstract":"For many organisms, there is agreement on the specific genomic region used for developing barcode markers. With nematodes, however, it has been found that the COI region designated for most animals lacks the taxonomic coverage (ability to amplify a diverse group of taxa) required of a metabarcoding marker. For that reason, studies on metabarcoding of nematodes thus far have utilized primarily regions within the highly conserved 18S ribosomal DNA. Two popular markers within this region are the ones flanked by the primer pairs NF1-18Sr2b and SSUF04-SSUR22. The NF1-18Sr2b primer pair, especially, has been critiqued as not being specific enough for nematodes leading to suggestions for other candidate markers while the SSUF04-SSUR22 region has hardly been tested on soil nematodes. The current study aimed to evaluate these two markers against other alternative ones within the 28S rDNA and the COI region for their suitability for nematode metabarcoding. The results showed that the NF1-18Sr2b marker could offer wide coverage and good resolution for characterizing soil nematodes. Sufficient availability of reference sequences for this region was found to be a significant factor that resulted in this marker outperforming the other markers, particularly the 18S-based SSUFO4-SSUR22 marker. None of the other tested regions compared with this marker in terms of the proportion of the taxa recovered. The COI-based marker had the lowest number of taxa recovered, and this was due to the poor performance of its primers and the insufficient number of reference sequences in public databases. In summary, this study highlights how dependent the success of metabarcoding is on the availability of a good reference sequence collection for the marker of choice as well as its taxonomic coverage.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44376162","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"DNA metabarcoding of Neotropical ichthyoplankton: Enabling high accuracy with lower cost","authors":"A. Nobile, D. Freitas‐Souza, Francisco J. Ruiz-Ruano, M. Nobile, G. O. D. Costa, F. P. Lima, J. Camacho, F. Foresti, Cláudio Oliveira","doi":"10.3897/mbmg.3.35060","DOIUrl":"https://doi.org/10.3897/mbmg.3.35060","url":null,"abstract":"Knowledge of ichthyoplankton dynamics is extremely important for conservation management as it can provide information about preferential spawning sites, reproductive period, migratory routes and recruitment success, which can be used to guide management and conservation efforts. However, identification of the eggs and larvae of Neotropical freshwater fish is a difficult task. DNA barcodes have emerged as an alternative and highly accurate approach for species identification, but DNA barcoding can be time-consuming and costly. To solve this problem, we aimed to develop a simple protocol based on DNA metabarcoding, to investigate whether it is possible to detect and quantify all species present in a pool of organisms. To do this, 230 larvae were cut in half, one half was sequenced by the Sanger technique and the other half was used to compose six arrays with a pool of larvae that were sequenced using a next-generation technique (NGS). The results of the Sanger sequencing allowed the identification of almost all larvae at species level, and the results from NGS showed high accuracy in species detection, ranging from 83% to 100%, with an average of 95% in all samples. No false positives were detected. The frequency of organisms in the two methods was positively correlated (Pearson), with low variation among species. In conclusion, this protocol represents a considerable advance in ichthyoplankton studies, allowing a rapid, cost-effective, quali-quantitative approach that improves the accuracy of identification.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-05-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43819240","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}