Laure Van den Bulcke, Annelies De Backer, B. Ampe, S. Maes, J. Wittoeck, W. Waegeman, K. Hostens, S. Derycke
DNA-based monitoring methods are potentially faster and cheaper compared to traditional morphological benthic identification. DNA metabarcoding involves various methodological choices which can introduce bias leading to a different outcome in biodiversity patterns. Therefore, it is important to harmonize DNA metabarcoding protocols to allow comparison across studies and this requires a good understanding of the effect of methodological choices on diversity estimates. This study investigated the impact of DNA and PCR replicates on the detection of macrobenthos species in locations with high, medium and low diversity. Our results show that two to three DNA replicates were needed in locations with a high and medium diversity to detect at least 80% of the species found in the six DNA replicates, while three to four replicates were needed in the location with low diversity. In contrast to general belief, larger body size or higher abundance of the species in a sample did not increase its detection prevalence among DNA replicates. However, rare species were less consistently detected across all DNA replicates of the location with high diversity compared to locations with less diversity. Our results further show that pooling of DNA replicates did not significantly alter diversity patterns, although a small number of rare species was lost. Finally, our results confirm high variation in species detection between PCR replicates, especially for the detection of rare species. These results contribute to create reliable, time and cost efficient metabarcoding protocols for the characterization of macrobenthos.
{"title":"Towards harmonization of DNA metabarcoding for monitoring marine macrobenthos: the effect of technical replicates and pooled DNA extractions on species detection","authors":"Laure Van den Bulcke, Annelies De Backer, B. Ampe, S. Maes, J. Wittoeck, W. Waegeman, K. Hostens, S. Derycke","doi":"10.3897/mbmg.5.71107","DOIUrl":"https://doi.org/10.3897/mbmg.5.71107","url":null,"abstract":"DNA-based monitoring methods are potentially faster and cheaper compared to traditional morphological benthic identification. DNA metabarcoding involves various methodological choices which can introduce bias leading to a different outcome in biodiversity patterns. Therefore, it is important to harmonize DNA metabarcoding protocols to allow comparison across studies and this requires a good understanding of the effect of methodological choices on diversity estimates. This study investigated the impact of DNA and PCR replicates on the detection of macrobenthos species in locations with high, medium and low diversity. Our results show that two to three DNA replicates were needed in locations with a high and medium diversity to detect at least 80% of the species found in the six DNA replicates, while three to four replicates were needed in the location with low diversity. In contrast to general belief, larger body size or higher abundance of the species in a sample did not increase its detection prevalence among DNA replicates. However, rare species were less consistently detected across all DNA replicates of the location with high diversity compared to locations with less diversity. Our results further show that pooling of DNA replicates did not significantly alter diversity patterns, although a small number of rare species was lost. Finally, our results confirm high variation in species detection between PCR replicates, especially for the detection of rare species. These results contribute to create reliable, time and cost efficient metabarcoding protocols for the characterization of macrobenthos.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-12-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47930681","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}
B. R. Leite, Pedro E. Vieira, J. Troncoso, F. Costa
DNA metabarcoding has great potential to improve marine biomonitoring programs by providing a rapid and accurate assessment of species composition in zoobenthic communities. However, some methodological improvements are still required, especially regarding failed detections, primers efficiency and incompleteness of databases. Here we assessed the efficiency of two different marker loci (COI and 18S) and three primer pairs in marine species detection through DNA metabarcoding of the macrozoobenthic communities colonizing three types of artificial substrates (slate, PVC and granite), sampled between 3 and 15 months of deployment. To accurately compare detection success between markers, we also compared the representativeness of the detected species in public databases and revised the reliability of the taxonomic assignments. Globally, we recorded extensive complementarity in the species detected by each marker, with 69% of the species exclusively detected by either 18S or COI. Individually, each of the three primer pairs recovered, at most, 52% of all species detected on the samples, showing also different abilities to amplify specific taxonomic groups. Most of the detected species have reliable reference sequences in their respective databases (82% for COI and 72% for 18S), meaning that when a species was detected by one marker and not by the other, it was most likely due to faulty amplification, and not by lack of matching sequences in the database. Overall, results showed the impact of marker and primer applied on species detection ability and indicated that, currently, if only a single marker or primer pair is employed in marine zoobenthos metabarcoding, a fair portion of the diversity may be overlooked.
{"title":"Comparing species detection success between molecular markers in DNA metabarcoding of coastal macroinvertebrates","authors":"B. R. Leite, Pedro E. Vieira, J. Troncoso, F. Costa","doi":"10.3897/mbmg.5.70063","DOIUrl":"https://doi.org/10.3897/mbmg.5.70063","url":null,"abstract":"DNA metabarcoding has great potential to improve marine biomonitoring programs by providing a rapid and accurate assessment of species composition in zoobenthic communities. However, some methodological improvements are still required, especially regarding failed detections, primers efficiency and incompleteness of databases. Here we assessed the efficiency of two different marker loci (COI and 18S) and three primer pairs in marine species detection through DNA metabarcoding of the macrozoobenthic communities colonizing three types of artificial substrates (slate, PVC and granite), sampled between 3 and 15 months of deployment. To accurately compare detection success between markers, we also compared the representativeness of the detected species in public databases and revised the reliability of the taxonomic assignments. Globally, we recorded extensive complementarity in the species detected by each marker, with 69% of the species exclusively detected by either 18S or COI. Individually, each of the three primer pairs recovered, at most, 52% of all species detected on the samples, showing also different abilities to amplify specific taxonomic groups. Most of the detected species have reliable reference sequences in their respective databases (82% for COI and 72% for 18S), meaning that when a species was detected by one marker and not by the other, it was most likely due to faulty amplification, and not by lack of matching sequences in the database. Overall, results showed the impact of marker and primer applied on species detection ability and indicated that, currently, if only a single marker or primer pair is employed in marine zoobenthos metabarcoding, a fair portion of the diversity may be overlooked.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-12-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42220789","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}
Liliane Boukhdoud, Carole Saliba, Lillian D. Parker, N. Mcinerney, Rhea Kahale, I. Saliba, J. Maldonado, M. B. D. Kharrat
Longevity of species populations depends largely on interactions among animals and plants in an ecosystem. Predation and seed dispersal are among the most important interactions necessary for species conservation and persistence, and diet analysis is a prerequisite tool to evaluate these interactions. Understanding these processes is crucial for identifying conservation targets and for executing efficient reforestation and ecological restoration. In this study, we applied a scat DNA metabarcoding technique using the P6-loop of the trnL (UAA) chloroplastic marker to describe the seasonal plant diet composition of 15 mammal species from a highly biodiverse Lebanese forest in the Eastern Mediterranean. We also recovered plant seeds, when present, from the scats for identification. The mammal species belong to 10 families from 5 different orders. More than 133 plant species from 54 plant families were detected and identified. Species from the Rosaceae, Poaceae, Apiaceae, Fabaceae, Fagaceae and Berberidaceae families were consumed by the majority of the mammals and should be taken into consideration in future reforestation and conservation projects. Our results showed that the DNA metabarcoding approach provides a promising method for tracking the dietary plant components of a wide diversity of mammals, yielding key insights into plant-animal interactions inside Lebanon’s forests.
{"title":"Using DNA metabarcoding to decipher the diet plant component of mammals from the Eastern Mediterranean region","authors":"Liliane Boukhdoud, Carole Saliba, Lillian D. Parker, N. Mcinerney, Rhea Kahale, I. Saliba, J. Maldonado, M. B. D. Kharrat","doi":"10.3897/mbmg.5.70107","DOIUrl":"https://doi.org/10.3897/mbmg.5.70107","url":null,"abstract":"Longevity of species populations depends largely on interactions among animals and plants in an ecosystem. Predation and seed dispersal are among the most important interactions necessary for species conservation and persistence, and diet analysis is a prerequisite tool to evaluate these interactions. Understanding these processes is crucial for identifying conservation targets and for executing efficient reforestation and ecological restoration. In this study, we applied a scat DNA metabarcoding technique using the P6-loop of the trnL (UAA) chloroplastic marker to describe the seasonal plant diet composition of 15 mammal species from a highly biodiverse Lebanese forest in the Eastern Mediterranean. We also recovered plant seeds, when present, from the scats for identification. The mammal species belong to 10 families from 5 different orders. More than 133 plant species from 54 plant families were detected and identified. Species from the Rosaceae, Poaceae, Apiaceae, Fabaceae, Fagaceae and Berberidaceae families were consumed by the majority of the mammals and should be taken into consideration in future reforestation and conservation projects. Our results showed that the DNA metabarcoding approach provides a promising method for tracking the dietary plant components of a wide diversity of mammals, yielding key insights into plant-animal interactions inside Lebanon’s forests.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49539540","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}
Alexis Canino, A. Bouchez, C. Laplace-Treyture, I. Domaizon, F. Rimet
Methods for biomonitoring of freshwater phytoplankton are evolving rapidly with eDNA-based methods, offering great complementarity with microscopy. Metabarcoding approaches have been more commonly used over the last years, with a continuous increase in the amount of data generated. Depending on the researchers and the way they assigned barcodes to species (bioinformatic pipelines and molecular reference databases), the taxonomic assignment obtained for HTS DNA reads might vary. This is also true for traditional taxonomic studies by microscopy with regular adjustments of the classification and taxonomy. For those reasons (leading to non-homogeneous taxonomies), gap-analyses and comparisons between studies become even more challenging and the curation processes to find potential consensus names are time-consuming. Here, we present a web-based application (Phytool), developed with ShinyApp (Rstudio), that aims to make the harmonisation of taxonomy easier and in a more efficient way, using a complete and up-to-date taxonomy reference database for freshwater microalgae. Phytool allows users to homogenise and update freshwater phytoplankton taxonomical names from sequence files and data tables directly uploaded in the application. It also gathers barcodes from curated references in a user-friendly way in which it is possible to search for specific organisms. All the data provided are downloadable with the possibility to apply filters in order to select only the required taxa and fields (e.g. specific taxonomic ranks). The main goal is to make accessible to a broad range of users the connection between microscopy and molecular biology and taxonomy through different ready-to-use functions. This study estimates that only 25% of species of freshwater phytoplankton in Phytobs are associated with a barcode. We plead for an increased effort to enrich reference databases by coupling taxonomy and molecular methods. Phytool should make this crucial work more efficient. The application is available at https://caninuzzo.shinyapps.io/phytool_v1/
{"title":"Phytool, a ShinyApp to homogenise taxonomy of freshwater microalgae from DNA barcodes and microscopic observations","authors":"Alexis Canino, A. Bouchez, C. Laplace-Treyture, I. Domaizon, F. Rimet","doi":"10.3897/mbmg.5.74096","DOIUrl":"https://doi.org/10.3897/mbmg.5.74096","url":null,"abstract":"Methods for biomonitoring of freshwater phytoplankton are evolving rapidly with eDNA-based methods, offering great complementarity with microscopy. Metabarcoding approaches have been more commonly used over the last years, with a continuous increase in the amount of data generated. Depending on the researchers and the way they assigned barcodes to species (bioinformatic pipelines and molecular reference databases), the taxonomic assignment obtained for HTS DNA reads might vary. This is also true for traditional taxonomic studies by microscopy with regular adjustments of the classification and taxonomy.\u0000 For those reasons (leading to non-homogeneous taxonomies), gap-analyses and comparisons between studies become even more challenging and the curation processes to find potential consensus names are time-consuming. Here, we present a web-based application (Phytool), developed with ShinyApp (Rstudio), that aims to make the harmonisation of taxonomy easier and in a more efficient way, using a complete and up-to-date taxonomy reference database for freshwater microalgae. Phytool allows users to homogenise and update freshwater phytoplankton taxonomical names from sequence files and data tables directly uploaded in the application. It also gathers barcodes from curated references in a user-friendly way in which it is possible to search for specific organisms. All the data provided are downloadable with the possibility to apply filters in order to select only the required taxa and fields (e.g. specific taxonomic ranks). The main goal is to make accessible to a broad range of users the connection between microscopy and molecular biology and taxonomy through different ready-to-use functions. This study estimates that only 25% of species of freshwater phytoplankton in Phytobs are associated with a barcode. We plead for an increased effort to enrich reference databases by coupling taxonomy and molecular methods. Phytool should make this crucial work more efficient.\u0000 The application is available at https://caninuzzo.shinyapps.io/phytool_v1/","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-12-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47564664","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}
Sirje Sildever, P. Laas, N. Kolesova, I. Lips, U. Lips, S. Nagai
Metabarcoding in combination with high-throughput sequencing (HTS) allows simultaneous detection of multiple taxa by targeting single or several taxonomically informative gene regions from environmental DNA samples. In this study, a multiple-marker HTS approach was applied to investigate the plankton diversity and seasonal succession in the Baltic Sea from winter to autumn. Four different markers targeting the 16S, 18S, and 28S ribosomal RNA genes were employed, including a marker for more efficient dinoflagellate detection. Typical seasonal changes were observed in phyto- and bacterioplankton communities. In phytoplankton, the appearance patterns of selected common, dominant, or harmful species followed the patterns also confirmed based on 20 years of phytoplankton monitoring data. In the case of zooplankton, both macro- and microzooplankton species were detected. However, no seasonal patterns were detected in their appearance. In total, 15 and 2 new zoo- and phytoplankton species were detected from the Baltic Sea. HTS approach was especially useful for detecting microzooplankton species as well as for investigating the co-occurrence and potential interactions of different taxa. The results of this study further exemplify the efficiency of metabarcoding for biodiversity monitoring and the advantage of employing multiple markers through the detection of species not identifiable based on a single marker survey and/or by traditional morphology-based methods.
{"title":"Plankton biodiversity and species co-occurrence based on environmental DNA – a multiple marker study","authors":"Sirje Sildever, P. Laas, N. Kolesova, I. Lips, U. Lips, S. Nagai","doi":"10.3897/mbmg.5.72371","DOIUrl":"https://doi.org/10.3897/mbmg.5.72371","url":null,"abstract":"Metabarcoding in combination with high-throughput sequencing (HTS) allows simultaneous detection of multiple taxa by targeting single or several taxonomically informative gene regions from environmental DNA samples. In this study, a multiple-marker HTS approach was applied to investigate the plankton diversity and seasonal succession in the Baltic Sea from winter to autumn. Four different markers targeting the 16S, 18S, and 28S ribosomal RNA genes were employed, including a marker for more efficient dinoflagellate detection. Typical seasonal changes were observed in phyto- and bacterioplankton communities. In phytoplankton, the appearance patterns of selected common, dominant, or harmful species followed the patterns also confirmed based on 20 years of phytoplankton monitoring data. In the case of zooplankton, both macro- and microzooplankton species were detected. However, no seasonal patterns were detected in their appearance. In total, 15 and 2 new zoo- and phytoplankton species were detected from the Baltic Sea. HTS approach was especially useful for detecting microzooplankton species as well as for investigating the co-occurrence and potential interactions of different taxa. The results of this study further exemplify the efficiency of metabarcoding for biodiversity monitoring and the advantage of employing multiple markers through the detection of species not identifiable based on a single marker survey and/or by traditional morphology-based methods.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-11-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48105690","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}
Haris Zafeiropoulos, L. Gargan, Sanni Hintikka, C. Pavloudi, J. Carlsson
The mitochondrial cytochrome C oxidase subunit I gene (COI) is commonly used in environmental DNA (eDNA) metabarcoding studies, especially for assessing metazoan diversity. Yet, a great number of COI operational taxonomic units (OTUs) or/and amplicon sequence variants (ASVs) retrieved from such studies do not get a taxonomic assignment with a reference sequence. To assess and investigate such sequences, we have developed the Dark mAtteR iNvestigator (DARN) software tool. For this purpose, a reference COI-oriented phylogenetic tree was built from 1,593 consensus sequences covering all the three domains of life. With respect to eukaryotes, consensus sequences at the family level were constructed from 183,330 sequences retrieved from the Midori reference 2 database, which represented 70% of the initial number of reference sequences. Similarly, sequences from 431 bacterial and 15 archaeal taxa at the family level (29% and 1% of the initial number of reference sequences respectively) were retrieved from the BOLD and the PFam databases. DARN makes use of this phylogenetic tree to investigate COI pre-processed sequences of amplicon samples to provide both a tabular and a graphical overview of their phylogenetic assignments. To evaluate DARN, both environmental and bulk metabarcoding samples from different aquatic environments using various primer sets were analysed. We demonstrate that a large proportion of non-target prokaryotic organisms, such as bacteria and archaea, are also amplified in eDNA samples and we suggest prokaryotic COI sequences to be included in the reference databases used for the taxonomy assignment to allow for further analyses of dark matter. DARN source code is available on GitHub at https://github.com/hariszaf/darn and as a Docker image at https://hub.docker.com/r/hariszaf/darn.
{"title":"The Dark mAtteR iNvestigator (DARN) tool: getting to know the known unknowns in COI amplicon data","authors":"Haris Zafeiropoulos, L. Gargan, Sanni Hintikka, C. Pavloudi, J. Carlsson","doi":"10.3897/mbmg.5.69657","DOIUrl":"https://doi.org/10.3897/mbmg.5.69657","url":null,"abstract":"The mitochondrial cytochrome C oxidase subunit I gene (COI) is commonly used in environmental DNA (eDNA) metabarcoding studies, especially for assessing metazoan diversity. Yet, a great number of COI operational taxonomic units (OTUs) or/and amplicon sequence variants (ASVs) retrieved from such studies do not get a taxonomic assignment with a reference sequence. To assess and investigate such sequences, we have developed the Dark mAtteR iNvestigator (DARN) software tool. For this purpose, a reference COI-oriented phylogenetic tree was built from 1,593 consensus sequences covering all the three domains of life. With respect to eukaryotes, consensus sequences at the family level were constructed from 183,330 sequences retrieved from the Midori reference 2 database, which represented 70% of the initial number of reference sequences. Similarly, sequences from 431 bacterial and 15 archaeal taxa at the family level (29% and 1% of the initial number of reference sequences respectively) were retrieved from the BOLD and the PFam databases. DARN makes use of this phylogenetic tree to investigate COI pre-processed sequences of amplicon samples to provide both a tabular and a graphical overview of their phylogenetic assignments. To evaluate DARN, both environmental and bulk metabarcoding samples from different aquatic environments using various primer sets were analysed. We demonstrate that a large proportion of non-target prokaryotic organisms, such as bacteria and archaea, are also amplified in eDNA samples and we suggest prokaryotic COI sequences to be included in the reference databases used for the taxonomy assignment to allow for further analyses of dark matter. DARN source code is available on GitHub at https://github.com/hariszaf/darn and as a Docker image at https://hub.docker.com/r/hariszaf/darn.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-11-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46747846","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}
Melissa R. Ingala, I. Werner, Allison M. Fitzgerald, Eugenia Naro‐Maciel
Characterising and monitoring biological diversity to foster sustainable ecosystems is highly recommended as urban centres rapidly expand. However, much of New York City’s biodiversity remains undescribed, including in the historically degraded, but recovering Bronx River Estuary. In a pilot study to identify organisms and characterise biodiversity patterns there, 18S rRNA gene amplicons (V1–V3 region), obtained from river sediments and surface waters of Hunts Point Riverside and Soundview Parks, were sequenced. Across 48 environmental samples collected over three seasons in 2015 and 2016, following quality control and contaminant removal, 2,763 Amplicon Sequence Variants (ASVs) were identified from 1,918,463 sequences. Rarefaction analysis showed sufficient sampling depth, and community composition varied over time and by substrate at the study sites over the sampling period. Protists, plants, fungi and animals, including organisms of management concern, such as Eastern oysters (Crassostrea virginica), wildlife pathogens and groups related to Harmful Algal Blooms, were detected. The most common taxa identified in river sediments were annelid worms, nematodes and diatoms. In the water column, the most commonly observed organisms were diatoms, algae of the phylum Cryptophyceae, ciliates and dinoflagellates. The presented dataset demonstrates the reach of 18S rRNA metabarcoding for characterising biodiversity in an urban estuary.
{"title":"18S rRNA amplicon sequence data (V1–V3) of the Bronx river estuary, New York","authors":"Melissa R. Ingala, I. Werner, Allison M. Fitzgerald, Eugenia Naro‐Maciel","doi":"10.3897/mbmg.5.69691","DOIUrl":"https://doi.org/10.3897/mbmg.5.69691","url":null,"abstract":"Characterising and monitoring biological diversity to foster sustainable ecosystems is highly recommended as urban centres rapidly expand. However, much of New York City’s biodiversity remains undescribed, including in the historically degraded, but recovering Bronx River Estuary. In a pilot study to identify organisms and characterise biodiversity patterns there, 18S rRNA gene amplicons (V1–V3 region), obtained from river sediments and surface waters of Hunts Point Riverside and Soundview Parks, were sequenced. Across 48 environmental samples collected over three seasons in 2015 and 2016, following quality control and contaminant removal, 2,763 Amplicon Sequence Variants (ASVs) were identified from 1,918,463 sequences. Rarefaction analysis showed sufficient sampling depth, and community composition varied over time and by substrate at the study sites over the sampling period. Protists, plants, fungi and animals, including organisms of management concern, such as Eastern oysters (Crassostrea virginica), wildlife pathogens and groups related to Harmful Algal Blooms, were detected. The most common taxa identified in river sediments were annelid worms, nematodes and diatoms. In the water column, the most commonly observed organisms were diatoms, algae of the phylum Cryptophyceae, ciliates and dinoflagellates. The presented dataset demonstrates the reach of 18S rRNA metabarcoding for characterising biodiversity in an urban estuary.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42732596","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}
The unprecedented ongoing biodiversity decline necessitates scalable means of monitoring in order to fully understand the underlying causes. DNA metabarcoding has the potential to provide a powerful tool for accurate and rapid biodiversity monitoring. Unfortunately, in many cases, a lack of universal standards undermines the widespread application of metabarcoding. One of the most important considerations in metabarcoding of plants, aside from selecting a potent barcode marker, is primer choice. Our study evaluates published ITS primers in silico and in vitro, through mock communities and presents newly designed primers. We were able to show that a large proportion of previously available ITS primers have unfavourable attributes. Our combined results support the recommendation of the introduced primers ITS-3p62plF1 and ITS-4unR1 as the best current universal plant specific ITS2 primer combination. We also found that PCR optimisation, such as the addition of 5% DMSO, is essential to obtain meaningful results in ITS2 metabarcoding. Finally, we conclude that continuous quality assurance is indispensable for reliable metabarcoding results.
{"title":"Internal transcribed spacer primer evaluation for vascular plant metabarcoding","authors":"Andreas Kolter, B. Gemeinholzer","doi":"10.3897/mbmg.5.68155","DOIUrl":"https://doi.org/10.3897/mbmg.5.68155","url":null,"abstract":"The unprecedented ongoing biodiversity decline necessitates scalable means of monitoring in order to fully understand the underlying causes. DNA metabarcoding has the potential to provide a powerful tool for accurate and rapid biodiversity monitoring. Unfortunately, in many cases, a lack of universal standards undermines the widespread application of metabarcoding. One of the most important considerations in metabarcoding of plants, aside from selecting a potent barcode marker, is primer choice. Our study evaluates published ITS primers in silico and in vitro, through mock communities and presents newly designed primers. We were able to show that a large proportion of previously available ITS primers have unfavourable attributes. Our combined results support the recommendation of the introduced primers ITS-3p62plF1 and ITS-4unR1 as the best current universal plant specific ITS2 primer combination. We also found that PCR optimisation, such as the addition of 5% DMSO, is essential to obtain meaningful results in ITS2 metabarcoding. Finally, we conclude that continuous quality assurance is indispensable for reliable metabarcoding results.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49207706","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}
Christy Meredith, Joel Hoffman, Anett Trebitz, Erik Pilgrim, Sarah Okum, John Martinson, Ellen S Cameron
For DNA metabarcoding to attain its potential as a community assessment tool, we need to better understand its performance versus traditional morphological identification and work to address any remaining performance gaps in incorporating DNA metabarcoding into community assessments. Using fragments of the 18S nuclear and 16S mitochondrial rRNA genes and two fragments of the mitochondrial COI marker, we examined the use of DNA metabarcoding and traditional morphological identification for understanding the diversity and composition of crustacean zooplankton at 42 sites across western Lake Superior. We identified 51 zooplankton taxa (genus or species, depending on the finest resolution of the taxon across all identification methods), of which 17 were identified using only morphological traits, 13 using only DNA and 21 using both methods. The taxa found using only DNA metabarcoding included four species and one genus-level identification not previously known to occur in Lake Superior, the presence of which still needs to be confirmed. A substantial portion of taxa that were identified to genus or species by morphological identification, but not identified using DNA metabarcoding, had zero ("no record") or ≤ 2 ("underrepresented records") reference barcodes in the BOLD or NCBI databases (63% for COI, 80% for 16S, 74% for 18S). The two COI marker fragments identified the most genus- and species-level taxa, whereas 18S was the only marker whose family-level percent sequence abundance patterns showed high correlation to composition patterns from morphological identification, based on a NMDS analysis of Bray-Curtis similarities. Multiple replicates were collected at a subset of sites and an occupancy analysis was performed, which indicated that rare taxa were more likely to be detected using DNA metabarcoding than traditional morphology. Our results support that DNA metabarcoding can augment morphological identification for estimating zooplankton diversity and composition of zooplankton over space and time, but may require use of multiple markers. Further addition of taxa to reference DNA databases will improve our ability to use DNA metabarcoding to identify zooplankton and other invertebrates in aquatic surveys.
{"title":"Evaluating the performance of DNA metabarcoding for assessment of zooplankton communities in Western Lake Superior using multiple markers.","authors":"Christy Meredith, Joel Hoffman, Anett Trebitz, Erik Pilgrim, Sarah Okum, John Martinson, Ellen S Cameron","doi":"10.3897/mbmg.5.64735","DOIUrl":"10.3897/mbmg.5.64735","url":null,"abstract":"<p><p>For DNA metabarcoding to attain its potential as a community assessment tool, we need to better understand its performance versus traditional morphological identification and work to address any remaining performance gaps in incorporating DNA metabarcoding into community assessments. Using fragments of the 18S nuclear and 16S mitochondrial rRNA genes and two fragments of the mitochondrial COI marker, we examined the use of DNA metabarcoding and traditional morphological identification for understanding the diversity and composition of crustacean zooplankton at 42 sites across western Lake Superior. We identified 51 zooplankton taxa (genus or species, depending on the finest resolution of the taxon across all identification methods), of which 17 were identified using only morphological traits, 13 using only DNA and 21 using both methods. The taxa found using only DNA metabarcoding included four species and one genus-level identification not previously known to occur in Lake Superior, the presence of which still needs to be confirmed. A substantial portion of taxa that were identified to genus or species by morphological identification, but not identified using DNA metabarcoding, had zero (\"no record\") or ≤ 2 (\"underrepresented records\") reference barcodes in the BOLD or NCBI databases (63% for COI, 80% for 16S, 74% for 18S). The two COI marker fragments identified the most genus- and species-level taxa, whereas 18S was the only marker whose family-level percent sequence abundance patterns showed high correlation to composition patterns from morphological identification, based on a NMDS analysis of Bray-Curtis similarities. Multiple replicates were collected at a subset of sites and an occupancy analysis was performed, which indicated that rare taxa were more likely to be detected using DNA metabarcoding than traditional morphology. Our results support that DNA metabarcoding can augment morphological identification for estimating zooplankton diversity and composition of zooplankton over space and time, but may require use of multiple markers. Further addition of taxa to reference DNA databases will improve our ability to use DNA metabarcoding to identify zooplankton and other invertebrates in aquatic surveys.</p>","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":"50 ","pages":"83-97"},"PeriodicalIF":0.0,"publicationDate":"2021-07-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8384127/pdf/nihms-1730443.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39356621","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
F. Rimet, E. Aylagas, Á. Borja, A. Bouchez, Alexis Canino, C. Chauvin, T. Chonova, Fedor Čiampor Jr, F. Costa, B. Ferrari, R. Gastineau, C. Goulon, M. Gugger, M. Holzmann, R. Jahn, M. Kahlert, Wolf-Henning Kusber, C. Laplace-Treyture, F. Leese, F. Leliaert, D. Mann, F. Marchand, V. Méléder, J. Pawłowski, S. Rasconi, S. Rivera, R. Rougerie, M. Schweizer, R. Trobajo, V. Vasselon, R. Vivien, A. Weigand, A. Witkowski, J. Zimmermann, T. Ekrem
DNA barcoding and metabarcoding is increasingly used to effectively and precisely assess and monitor biodiversity in aquatic ecosystems. As these methods rely on data availability and quality of barcode reference libraries, it is important to develop and follow best practices to ensure optimal quality and traceability of the metadata associated with the reference barcodes used for identification. Sufficient metadata, as well as vouchers, corresponding to each reference barcode must be available to ensure reliable barcode library curation and, thereby, provide trustworthy baselines for downstream molecular species identification. This document (1) specifies the data and metadata required to ensure the relevance, the accessibility and traceability of DNA barcodes and (2) specifies the recommendations for DNA harvesting and for the storage of both voucher specimens/samples and barcode data.
{"title":"Metadata standards and practical guidelines for specimen and DNA curation when building barcode reference libraries for aquatic life","authors":"F. Rimet, E. Aylagas, Á. Borja, A. Bouchez, Alexis Canino, C. Chauvin, T. Chonova, Fedor Čiampor Jr, F. Costa, B. Ferrari, R. Gastineau, C. Goulon, M. Gugger, M. Holzmann, R. Jahn, M. Kahlert, Wolf-Henning Kusber, C. Laplace-Treyture, F. Leese, F. Leliaert, D. Mann, F. Marchand, V. Méléder, J. Pawłowski, S. Rasconi, S. Rivera, R. Rougerie, M. Schweizer, R. Trobajo, V. Vasselon, R. Vivien, A. Weigand, A. Witkowski, J. Zimmermann, T. Ekrem","doi":"10.3897/MBMG.5.58056","DOIUrl":"https://doi.org/10.3897/MBMG.5.58056","url":null,"abstract":"DNA barcoding and metabarcoding is increasingly used to effectively and precisely assess and monitor biodiversity in aquatic ecosystems. As these methods rely on data availability and quality of barcode reference libraries, it is important to develop and follow best practices to ensure optimal quality and traceability of the metadata associated with the reference barcodes used for identification. Sufficient metadata, as well as vouchers, corresponding to each reference barcode must be available to ensure reliable barcode library curation and, thereby, provide trustworthy baselines for downstream molecular species identification. This document (1) specifies the data and metadata required to ensure the relevance, the accessibility and traceability of DNA barcodes and (2) specifies the recommendations for DNA harvesting and for the storage of both voucher specimens/samples and barcode data.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46480139","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}
京公网安备 11010802042870号
京ICP备2023020795号-1
扫码关注我们
求助内容：
应助结果提醒方式：