Till-Hendrik Macher, Robin Schütz, Atakan Yildiz, A. Beermann, F. Leese
Environmental DNA (eDNA) metabarcoding has become a powerful tool for examining fish communities. Prior to the introduction of eDNA-based assessments into regulatory monitoring contexts (e.g., EU Water Framework Directive), there is a demand for methodological standardization. To ensure methodical accuracy and to meet regulatory standards, various sampling, laboratory and bioinformatic workflows have been established. However, a crucial prerequisite for comprehensive fish monitoring is the choice of suitable primer pairs to accurately identify the fishes present in a given water body. Various fish-specific primer pairs targeting different genetic marker regions were published over the past decade. However, a dedicated study to evaluate the performance of frequently applied fish primer pairs to assess Central European fish species has not yet been conducted. Therefore, we created an artificial 'mock' community composed of DNA from 45 Central European fish species and examined the detection ability and reproducibility of five primer pairs. Our study highlights the effect of primer choice and bioinformatic filtering on the outcome of eDNA metabarcoding results. From the five primer pairs evaluated in our study the tele02 (12S gene) primer pair was the best choice for eDNA metabarcoding of Central European freshwater fish. Also, the MiFish-U (12S) and SeaDNA-mid (COI) primer pairs displayed good detection ability and reproducibility. However, less specific primer pairs (i.e., targeting vertebrates) were found to be less reliable and generated high numbers of false-positive and false-negative detections. Our study illustrates how the careful selection of primer pairs and bioinformatic pipelines can make eDNA metabarcoding a more reliable tool for fish monitoring.
{"title":"Evaluating five primer pairs for environmental DNA metabarcoding of Central European fish species based on mock communities","authors":"Till-Hendrik Macher, Robin Schütz, Atakan Yildiz, A. Beermann, F. Leese","doi":"10.3897/mbmg.7.103856","DOIUrl":"https://doi.org/10.3897/mbmg.7.103856","url":null,"abstract":"Environmental DNA (eDNA) metabarcoding has become a powerful tool for examining fish communities. Prior to the introduction of eDNA-based assessments into regulatory monitoring contexts (e.g., EU Water Framework Directive), there is a demand for methodological standardization. To ensure methodical accuracy and to meet regulatory standards, various sampling, laboratory and bioinformatic workflows have been established. However, a crucial prerequisite for comprehensive fish monitoring is the choice of suitable primer pairs to accurately identify the fishes present in a given water body. Various fish-specific primer pairs targeting different genetic marker regions were published over the past decade. However, a dedicated study to evaluate the performance of frequently applied fish primer pairs to assess Central European fish species has not yet been conducted. Therefore, we created an artificial 'mock' community composed of DNA from 45 Central European fish species and examined the detection ability and reproducibility of five primer pairs. Our study highlights the effect of primer choice and bioinformatic filtering on the outcome of eDNA metabarcoding results. From the five primer pairs evaluated in our study the tele02 (12S gene) primer pair was the best choice for eDNA metabarcoding of Central European freshwater fish. Also, the MiFish-U (12S) and SeaDNA-mid (COI) primer pairs displayed good detection ability and reproducibility. However, less specific primer pairs (i.e., targeting vertebrates) were found to be less reliable and generated high numbers of false-positive and false-negative detections. Our study illustrates how the careful selection of primer pairs and bioinformatic pipelines can make eDNA metabarcoding a more reliable tool for fish monitoring.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45135912","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}
Bastien Parisy, N. Schmidt, Helena Wirta, Lærke Stewart, L. Pellissier, W. Holben, Samuel B Pannoni, P. Somervuo, Mirkka M. Jones, J. Siren, E. Vesterinen, O. Ovaskainen, T. Roslin
Understanding how different taxa respond to abiotic characteristics of the environment is of key interest for understanding the assembly of communities. Yet, whether eDNA data will suffice to accurately capture environmental imprints has been the topic of some debate. In this study, we characterised patterns of species occurrences and co-occurrences in Zackenberg in northeast Greenland using environmental DNA. To explore the potential for extracting ecological signals from eDNA data alone, we compared two approaches (visual vegetation surveys and soil eDNA metabarcoding) to describing plant communities and their responses to abiotic conditions. We then examined plant associations with microbes using a joint species distribution model. We found that most (68%) of plant genera were detectable by both vegetation surveys and eDNA signatures. Species-specific occurrence data revealed how plants, bacteria and fungi responded to their abiotic environment – with plants, bacteria and fungi all responding similarly to soil moisture. Nonetheless, a large proportion of fungi decreased in occurrences with increasing soil temperature. Regarding biotic associations, the nature and proportion of the plant-microbe associations detected were consistent between plant data identified via vegetation surveys and eDNA. Of pairs of plants and microbe genera showing statistically supported associations (while accounting for joint responses to the environment), plants and bacteria mainly showed negative associations, whereas plants and fungi mainly showed positive associations. Ample ecological signals detected by both vegetation surveys and by eDNA-based methods and a general correspondence in biotic associations inferred by both methods, suggested that purely eDNA-based approaches constitute a promising and easily applicable tool for studying plant-soil microbial associations in the Arctic and elsewhere.
{"title":"Ecological signals of arctic plant-microbe associations are consistent across eDNA and vegetation surveys","authors":"Bastien Parisy, N. Schmidt, Helena Wirta, Lærke Stewart, L. Pellissier, W. Holben, Samuel B Pannoni, P. Somervuo, Mirkka M. Jones, J. Siren, E. Vesterinen, O. Ovaskainen, T. Roslin","doi":"10.3897/mbmg.7.99979","DOIUrl":"https://doi.org/10.3897/mbmg.7.99979","url":null,"abstract":"Understanding how different taxa respond to abiotic characteristics of the environment is of key interest for understanding the assembly of communities. Yet, whether eDNA data will suffice to accurately capture environmental imprints has been the topic of some debate. In this study, we characterised patterns of species occurrences and co-occurrences in Zackenberg in northeast Greenland using environmental DNA. To explore the potential for extracting ecological signals from eDNA data alone, we compared two approaches (visual vegetation surveys and soil eDNA metabarcoding) to describing plant communities and their responses to abiotic conditions. We then examined plant associations with microbes using a joint species distribution model. We found that most (68%) of plant genera were detectable by both vegetation surveys and eDNA signatures. Species-specific occurrence data revealed how plants, bacteria and fungi responded to their abiotic environment – with plants, bacteria and fungi all responding similarly to soil moisture. Nonetheless, a large proportion of fungi decreased in occurrences with increasing soil temperature. Regarding biotic associations, the nature and proportion of the plant-microbe associations detected were consistent between plant data identified via vegetation surveys and eDNA. Of pairs of plants and microbe genera showing statistically supported associations (while accounting for joint responses to the environment), plants and bacteria mainly showed negative associations, whereas plants and fungi mainly showed positive associations. Ample ecological signals detected by both vegetation surveys and by eDNA-based methods and a general correspondence in biotic associations inferred by both methods, suggested that purely eDNA-based approaches constitute a promising and easily applicable tool for studying plant-soil microbial associations in the Arctic and elsewhere.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47128624","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}
A. Hubancheva, Vedran Bozicevic, J. Morinière, Holger R. Goerlitz
A comprehensive understanding of trophic interactions in terrestrial ecosystems is crucial for ecological research and conservation. Recent advances in non-invasive methods, such as environmental DNA (eDNA) metabarcoding, have enabled researchers to collect vast amounts of data on wild animal diets. However, sharing this data and metadata effectively and transparently presents new challenges. To address this, a new type of scholarly journal publication has emerged that aims to describe datasets rather than report research investigations. In this paper, we present a dataset of consumed prey species and parasites based on the metabarcoding of 113 faecal samples from the greater and lesser mouse-eared bats (Myotis myotis and Myotis blythii), along with a detailed description of the data sampling, laboratory analysis, and bioinformatics pipeline. Our dataset comprises 1018 unique Barcode Index Numbers (BINs) from 12 Classes and 43 Orders. In addition, we provide interactive Krona charts to visually summarize the taxonomic relationships and relative read abundance of the consumed prey species and parasites. This data can be used for meta-analysis, exploring new predator-prey and host-parasite interactions, studying inter- and intraspecific ecological interactions, and informing protected area management, among other applications. By sharing this dataset, we hope to encourage other researchers to use it to answer additional ecological questions and advance our understanding of trophic interactions in terrestrial ecosystems.
{"title":"DNA metabarcoding data from faecal samples of the lesser (Myotis blythii) and the greater (Myotis myotis) mouse-eared bats from Bulgaria","authors":"A. Hubancheva, Vedran Bozicevic, J. Morinière, Holger R. Goerlitz","doi":"10.3897/mbmg.7.106844","DOIUrl":"https://doi.org/10.3897/mbmg.7.106844","url":null,"abstract":"A comprehensive understanding of trophic interactions in terrestrial ecosystems is crucial for ecological research and conservation. Recent advances in non-invasive methods, such as environmental DNA (eDNA) metabarcoding, have enabled researchers to collect vast amounts of data on wild animal diets. However, sharing this data and metadata effectively and transparently presents new challenges. To address this, a new type of scholarly journal publication has emerged that aims to describe datasets rather than report research investigations. In this paper, we present a dataset of consumed prey species and parasites based on the metabarcoding of 113 faecal samples from the greater and lesser mouse-eared bats (Myotis myotis and Myotis blythii), along with a detailed description of the data sampling, laboratory analysis, and bioinformatics pipeline. Our dataset comprises 1018 unique Barcode Index Numbers (BINs) from 12 Classes and 43 Orders. In addition, we provide interactive Krona charts to visually summarize the taxonomic relationships and relative read abundance of the consumed prey species and parasites. This data can be used for meta-analysis, exploring new predator-prey and host-parasite interactions, studying inter- and intraspecific ecological interactions, and informing protected area management, among other applications. By sharing this dataset, we hope to encourage other researchers to use it to answer additional ecological questions and advance our understanding of trophic interactions in terrestrial ecosystems.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47284989","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}
Aquatic emergent insect communities form an important link between aquatic and terrestrial ecosystems, yet studying them is costly and time-consuming as they are usually diverse and superabundant. Metabarcoding is a valuable tool to investigate arthropod community compositions, however high-throughput applications, such as for biomonitoring, require cost-effective and user-friendly procedures. To investigate if the time-consuming and labour-intensive DNA extraction step can be omitted in metabarcoding, we studied the difference in detection rates and individual read abundance using standard DNA extraction versus direct PCR protocols. Metabarcoding with and without DNA extraction was performed with artificially created communities of known composition as well as on natural communities both of the dipteran family Chironomidae to compare detection rates, individual read abundances and presence-absence community composition. We found that the novel approach of direct PCR metabarcoding presented here did not alter detection rates and had a minor effect on individual read abundances in artificially created communities. Furthermore, presence-absence community compositions of natural chironomid communities were highly comparable using both approaches. In conclusion, we showed that direct PCR protocols can be applied in chironomid metabarcoding approaches, with possible application for a wider range of arthropod taxa, enabling us to study communities more efficiently in the future.
{"title":"Direct PCR meets high-throughput sequencing – metabarcoding of chironomid communities without DNA extraction","authors":"N. Röder, K. Schwenk","doi":"10.3897/mbmg.7.102455","DOIUrl":"https://doi.org/10.3897/mbmg.7.102455","url":null,"abstract":"Aquatic emergent insect communities form an important link between aquatic and terrestrial ecosystems, yet studying them is costly and time-consuming as they are usually diverse and superabundant. Metabarcoding is a valuable tool to investigate arthropod community compositions, however high-throughput applications, such as for biomonitoring, require cost-effective and user-friendly procedures. To investigate if the time-consuming and labour-intensive DNA extraction step can be omitted in metabarcoding, we studied the difference in detection rates and individual read abundance using standard DNA extraction versus direct PCR protocols. Metabarcoding with and without DNA extraction was performed with artificially created communities of known composition as well as on natural communities both of the dipteran family Chironomidae to compare detection rates, individual read abundances and presence-absence community composition. We found that the novel approach of direct PCR metabarcoding presented here did not alter detection rates and had a minor effect on individual read abundances in artificially created communities. Furthermore, presence-absence community compositions of natural chironomid communities were highly comparable using both approaches. In conclusion, we showed that direct PCR protocols can be applied in chironomid metabarcoding approaches, with possible application for a wider range of arthropod taxa, enabling us to study communities more efficiently in the future.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46608225","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}
Andreas Kolter, M. Husemann, L. Podsiadlowski, B. Gemeinholzer
Landscape changes, over time, lead to changes of floral resources available to pollinators, which in turn may result in the disappearance of ecologically specialized species. Here, we use pollen metabarcoding to infer historic and recent interactions between plants and bumblebees (Bombus). Bumblebees from Cuxhaven (Germany) were sampled from historical museum collections (1968/69) and in the field (2019). Pollen attached to their bodies was barcoded using multiple plant markers (ITS1, ITS2 and trnL-P6 loop). Our results show shifts in foraging habits between the historic and recent sampling periods, mostly determined by fewer Fabaceae interactions in 2019. The successful implementation of scalable molecular techniques for the analysis of historical pollen samples underscores the value of museum collections as a resource for biodiversity research. This study provides proof of concept of a comparative analysis of recent and historical pollination data. However, to ensure the robustness of our results, it is crucial to consider the broader methodology used. Our study found variation in the efficacy of the three plant barcoding markers. The ITS1 marker exhibited the highest species-level identification success, while the trnL-P6 loop demonstrated utility in amplifying degraded DNA across diverse plant families.
{"title":"Pollen metabarcoding of museum specimens and recently collected bumblebees (Bombus) indicates foraging shifts","authors":"Andreas Kolter, M. Husemann, L. Podsiadlowski, B. Gemeinholzer","doi":"10.3897/mbmg.7.86883","DOIUrl":"https://doi.org/10.3897/mbmg.7.86883","url":null,"abstract":"Landscape changes, over time, lead to changes of floral resources available to pollinators, which in turn may result in the disappearance of ecologically specialized species. Here, we use pollen metabarcoding to infer historic and recent interactions between plants and bumblebees (Bombus). Bumblebees from Cuxhaven (Germany) were sampled from historical museum collections (1968/69) and in the field (2019). Pollen attached to their bodies was barcoded using multiple plant markers (ITS1, ITS2 and trnL-P6 loop). Our results show shifts in foraging habits between the historic and recent sampling periods, mostly determined by fewer Fabaceae interactions in 2019. The successful implementation of scalable molecular techniques for the analysis of historical pollen samples underscores the value of museum collections as a resource for biodiversity research. This study provides proof of concept of a comparative analysis of recent and historical pollination data. However, to ensure the robustness of our results, it is crucial to consider the broader methodology used. Our study found variation in the efficacy of the three plant barcoding markers. The ITS1 marker exhibited the highest species-level identification success, while the trnL-P6 loop demonstrated utility in amplifying degraded DNA across diverse plant families.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45250837","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}
Mingxin Liu, C. Burridge, L. Clarke, S. Baker, G. Jordan
Estimating species biomass or abundance from the number of high-throughput sequencing (HTS) reads is an aspirational goal for DNA metabarcoding, yet studies have found varied correlations. Performance varies depending on the gene marker and taxonomic group and, in part, may be related to primer-template mismatches, which are likely to exhibit phylogenetic signals. In this study, we compared commonly used fragments of two gene markers for beetles, the mitochondrial cytochrome c oxidase subunit I (COI) and 16S ribosomal RNA (16S), which have similar lengths, but different propensity for primer-template mismatches. We tested whether primer-template mismatches influence the relationship between species biomass and HTS read abundance and whether the effect of mismatches was explained by phylogeny. A significant correlation between species biomass and HTS read abundance existed for 16S, but not for COI, which had more primer-template mismatches. Models incorporating the effects of mismatch type or number improved the estimation of species biomass from HTS read abundance for COI and strong phylogenetic signals were identified. Researchers seeking to quantify biomass from metabarcoding studies should consider the effect of primer-template mismatches for the taxonomic group of interest and, for beetles, 16S appears a good candidate. Phylogenetic correction can also improve biomass estimation when using gene markers with higher primer mismatching.
{"title":"Does phylogeny explain bias in quantitative DNA metabarcoding?","authors":"Mingxin Liu, C. Burridge, L. Clarke, S. Baker, G. Jordan","doi":"10.3897/mbmg.7.101266","DOIUrl":"https://doi.org/10.3897/mbmg.7.101266","url":null,"abstract":"Estimating species biomass or abundance from the number of high-throughput sequencing (HTS) reads is an aspirational goal for DNA metabarcoding, yet studies have found varied correlations. Performance varies depending on the gene marker and taxonomic group and, in part, may be related to primer-template mismatches, which are likely to exhibit phylogenetic signals. In this study, we compared commonly used fragments of two gene markers for beetles, the mitochondrial cytochrome c oxidase subunit I (COI) and 16S ribosomal RNA (16S), which have similar lengths, but different propensity for primer-template mismatches. We tested whether primer-template mismatches influence the relationship between species biomass and HTS read abundance and whether the effect of mismatches was explained by phylogeny. A significant correlation between species biomass and HTS read abundance existed for 16S, but not for COI, which had more primer-template mismatches. Models incorporating the effects of mismatch type or number improved the estimation of species biomass from HTS read abundance for COI and strong phylogenetic signals were identified. Researchers seeking to quantify biomass from metabarcoding studies should consider the effect of primer-template mismatches for the taxonomic group of interest and, for beetles, 16S appears a good candidate. Phylogenetic correction can also improve biomass estimation when using gene markers with higher primer mismatching.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-06-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45550648","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}
M. Okanishi, Hisanori Kohtsuka, Qianqian Wu, Junpei Shinji, Naoki Shibata, Takashi Tamada, T. Nakano, T. Minamoto
Brittle stars (class Ophiuroidea) are marine invertebrates comprising approximately 2,100 extant species, and are considered to constitute the most diverse taxon of the phylum Echinodermata. As a non-invasive method for monitoring biodiversity, we developed two new sets of PCR primers for metabarcoding environmental DNA (eDNA) from brittle stars. The new primer sets were designed to amplify 2 short regions of the mitochondrial 16S rRNA gene, comprising a conserved region (111–115 bp, 112 bp on average; named “16SOph1”) and a hyper-variable region (180–195 bp, 185 bp on average; named “16SOph2”) displaying interspecific variation. The performance of the primers was tested using eDNA obtained from two sources: a) rearing water of an 2.5 or 170 L aquarium tanks containing 15 brittle star species and b) from natural seawater collected around Misaki, the Pacific coast of central Japan, at depths ranging from shallow (2 m) to deep (> 200 m) sea. To build a reference library, we obtained 16S rRNA sequences of brittle star specimens collected from around Misaki and from similar depths in Japan, and sequences registered in International Nucleotide Sequence Database Collaboration. As a result of comparison of the obtained eDNA sequences with the reference library 37 (including cryptic species) and 26 brittle star species were detected with certain identities by 16SOph1 and 16SOph2 analyses, respectively. In shallow water, the number of species and reads other than the brittle stars detected with 16SOph1 was less than 10% of the total number. On the other hand, the number of brittle star species and reads detected with 16SOph2 was less than half of the total number, and the number of detected non-brittle star metazoan species ranged from 20 to 46 species across 6 to 8 phyla (only the reads at the “Tank” were less than 0.001%). The number of non-brittle star species and reads at 80 m was less than 10% with both of the primer sets. These findings suggest that 16SOph1 is specific to the brittle star and 16SOph2 is suitable for a variety of marine metazoans. It appears, however, that further optimization of primer sequences would still be necessary to avoid possible PCR dropouts from eDNA extracts. Moreover, a detailed elucidation of the brittle star fauna in the examined area, and the accurate identification of brittle star species in the current DNA databank is required.
{"title":"Development of two new sets of PCR primers for eDNA metabarcoding of brittle stars (Echinodermata, Ophiuroidea)","authors":"M. Okanishi, Hisanori Kohtsuka, Qianqian Wu, Junpei Shinji, Naoki Shibata, Takashi Tamada, T. Nakano, T. Minamoto","doi":"10.3897/mbmg.7.94298","DOIUrl":"https://doi.org/10.3897/mbmg.7.94298","url":null,"abstract":"Brittle stars (class Ophiuroidea) are marine invertebrates comprising approximately 2,100 extant species, and are considered to constitute the most diverse taxon of the phylum Echinodermata. As a non-invasive method for monitoring biodiversity, we developed two new sets of PCR primers for metabarcoding environmental DNA (eDNA) from brittle stars. The new primer sets were designed to amplify 2 short regions of the mitochondrial 16S rRNA gene, comprising a conserved region (111–115 bp, 112 bp on average; named “16SOph1”) and a hyper-variable region (180–195 bp, 185 bp on average; named “16SOph2”) displaying interspecific variation. The performance of the primers was tested using eDNA obtained from two sources: a) rearing water of an 2.5 or 170 L aquarium tanks containing 15 brittle star species and b) from natural seawater collected around Misaki, the Pacific coast of central Japan, at depths ranging from shallow (2 m) to deep (> 200 m) sea. To build a reference library, we obtained 16S rRNA sequences of brittle star specimens collected from around Misaki and from similar depths in Japan, and sequences registered in International Nucleotide Sequence Database Collaboration. As a result of comparison of the obtained eDNA sequences with the reference library 37 (including cryptic species) and 26 brittle star species were detected with certain identities by 16SOph1 and 16SOph2 analyses, respectively. In shallow water, the number of species and reads other than the brittle stars detected with 16SOph1 was less than 10% of the total number. On the other hand, the number of brittle star species and reads detected with 16SOph2 was less than half of the total number, and the number of detected non-brittle star metazoan species ranged from 20 to 46 species across 6 to 8 phyla (only the reads at the “Tank” were less than 0.001%). The number of non-brittle star species and reads at 80 m was less than 10% with both of the primer sets. These findings suggest that 16SOph1 is specific to the brittle star and 16SOph2 is suitable for a variety of marine metazoans. It appears, however, that further optimization of primer sequences would still be necessary to avoid possible PCR dropouts from eDNA extracts. Moreover, a detailed elucidation of the brittle star fauna in the examined area, and the accurate identification of brittle star species in the current DNA databank is required.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-02-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42626336","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}
Bourret, Audrey, Nozères, Claude, Parent, Eric, Parent, Geneviève J.
Biodiversity assessments relying on DNA have increased rapidly over the last decade. However, the reliability of taxonomic assignments in metabarcoding studies is variable and affected by the reference databases and the assignment methods used. Species level assignments are usually considered as reliable using regional libraries but unreliable using public repositories. In this study, we aimed to test this assumption for metazoan species detected in the Gulf of St. Lawrence in the Northwest Atlantic. We first created a regional library (GSL-rl) by data mining COI barcode sequences from BOLD, and included a reliability ranking system for species assignments. We then estimated 1) the accuracy and precision of the public repository NCBI-nt for species assignments using sequences from the regional library and 2) compared the detection and reliability of species assignments of a metabarcoding dataset using either NCBI-nt or the regional library and popular assignment methods. With NCBI-nt and sequences from the regional library, the BLAST-LCA (least common ancestor) method was the most precise method for species assignments, but the accuracy was higher with the BLAST-TopHit method (>80% over all taxa, between 70% and 90% amongst taxonomic groups). With the metabarcoding dataset, the reliability of species assignments was greater using GSL-rl compared to NCBI-nt. However, we also observed that the total number of reliable species assignments could be maximized using both GSL-rl and NCBI-nt with different optimized assignment methods. The use of a two-step approach for species assignments, i.e., using a regional library and a public repository, could improve the reliability and the number of detected species in metabarcoding studies.
过去十年来，依赖DNA的生物多样性评估迅速增加。然而，元条形码研究中分类分配的可靠性是可变的，并且受参考数据库和使用的分配方法的影响。物种水平的分配通常被认为是可靠的使用区域图书馆，但不可靠的使用公共库。在这项研究中，我们的目的是在西北大西洋的圣劳伦斯湾检测到的后生动物物种中验证这一假设。我们首先通过对BOLD中COI条形码序列的数据挖掘创建了一个区域库(GSL-rl)，并包含了一个物种分配的可靠性排序系统。然后，我们估计了1)公共数据库NCBI-nt使用来自区域库的序列进行物种分配的准确性和精密度;2)比较了使用NCBI-nt或区域库和流行的分配方法进行元条形码数据集物种分配的检测和可靠性。利用NCBI-nt和区域文库序列，BLAST-LCA (least common ancestor)方法是最精确的物种分配方法，但BLAST-TopHit方法的准确率更高(在所有分类群中为80%，在分类群中为70% ~ 90%)。与NCBI-nt相比，在元条形码数据集上，GSL-rl的物种分配可靠性更高。然而，我们也观察到，GSL-rl和NCBI-nt采用不同的优化分配方法都能最大限度地获得可靠的物种分配。采用区域文库和公共文库两步法进行物种分配，可以提高元条形码研究的可靠性和检测物种的数量。
{"title":"Maximizing the reliability and the number of species assignments in metabarcoding studies using a curated regional library and a public repository","authors":"Bourret, Audrey, Nozères, Claude, Parent, Eric, Parent, Geneviève J.","doi":"10.3897/mbmg.7.98539","DOIUrl":"https://doi.org/10.3897/mbmg.7.98539","url":null,"abstract":"Biodiversity assessments relying on DNA have increased rapidly over the last decade. However, the reliability of taxonomic assignments in metabarcoding studies is variable and affected by the reference databases and the assignment methods used. Species level assignments are usually considered as reliable using regional libraries but unreliable using public repositories. In this study, we aimed to test this assumption for metazoan species detected in the Gulf of St. Lawrence in the Northwest Atlantic. We first created a regional library (GSL-rl) by data mining COI barcode sequences from BOLD, and included a reliability ranking system for species assignments. We then estimated 1) the accuracy and precision of the public repository NCBI-nt for species assignments using sequences from the regional library and 2) compared the detection and reliability of species assignments of a metabarcoding dataset using either NCBI-nt or the regional library and popular assignment methods. With NCBI-nt and sequences from the regional library, the BLAST-LCA (least common ancestor) method was the most precise method for species assignments, but the accuracy was higher with the BLAST-TopHit method (>80% over all taxa, between 70% and 90% amongst taxonomic groups). With the metabarcoding dataset, the reliability of species assignments was greater using GSL-rl compared to NCBI-nt. However, we also observed that the total number of reliable species assignments could be maximized using both GSL-rl and NCBI-nt with different optimized assignment methods. The use of a two-step approach for species assignments, i.e., using a regional library and a public repository, could improve the reliability and the number of detected species in metabarcoding studies.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":"60 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-02-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"136173539","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}
Hailong Dou, Mi Wang, Xuwang Yin, Limin Feng, Haitao Yang
The Eurasian otter Lutra lutra is a generalist carnivore that is widely distributed in many aquatic ecosystems. Based on its inherent attributes of opportunistic foraging behaviour and broad dietary range, it is naturally considered a potential sampler of the diversity of aquatic vertebrates. To test the ability and efficiency of otters as a diversity sampler, we used DNA metabarcoding to investigate the composition in vertebrates of the diet of otters that inhabit a forest stream area in northeast China. Twenty vertebrate prey taxa were detected in 98 otter spraints. Otter diet mainly comprised aquatic fishes (59.4%) and amphibians (39.0%). We also used traditional approaches to investigate fish communities at 60 sampling sites in the same area to determine the relationship between fish population composition in the environment and otter diet. The comparison revealed that 28 species of fish were distributed in this area, of which five are simultaneously detected in otter spraints. This indicates that molecular analysis of the diet of otters is not an ideal approach for investigating fish diversity, at least when using the 12SV5 primer pair. Based on a review of the available molecular research on otter diet, we conclude that the low species resolution may be due to the presence of many closely-related prey species in native habitats and lack of suitable barcodes. Considering the remarkable power of diet metabarcoding analysis in capturing elusive and rare species, it represents an approach that can compensate for the defects associated with fishing methods and we suggest that it can be used as an auxiliary means of measuring traditional fish diversity.
{"title":"Can the Eurasian otter (Lutra lutra) be used as an effective sampler of fish diversity? Using molecular assessment of otter diet to survey fish communities","authors":"Hailong Dou, Mi Wang, Xuwang Yin, Limin Feng, Haitao Yang","doi":"10.3897/mbmg.7.96733","DOIUrl":"https://doi.org/10.3897/mbmg.7.96733","url":null,"abstract":"The Eurasian otter Lutra lutra is a generalist carnivore that is widely distributed in many aquatic ecosystems. Based on its inherent attributes of opportunistic foraging behaviour and broad dietary range, it is naturally considered a potential sampler of the diversity of aquatic vertebrates. To test the ability and efficiency of otters as a diversity sampler, we used DNA metabarcoding to investigate the composition in vertebrates of the diet of otters that inhabit a forest stream area in northeast China. Twenty vertebrate prey taxa were detected in 98 otter spraints. Otter diet mainly comprised aquatic fishes (59.4%) and amphibians (39.0%). We also used traditional approaches to investigate fish communities at 60 sampling sites in the same area to determine the relationship between fish population composition in the environment and otter diet. The comparison revealed that 28 species of fish were distributed in this area, of which five are simultaneously detected in otter spraints. This indicates that molecular analysis of the diet of otters is not an ideal approach for investigating fish diversity, at least when using the 12SV5 primer pair. Based on a review of the available molecular research on otter diet, we conclude that the low species resolution may be due to the presence of many closely-related prey species in native habitats and lack of suitable barcodes. Considering the remarkable power of diet metabarcoding analysis in capturing elusive and rare species, it represents an approach that can compensate for the defects associated with fishing methods and we suggest that it can be used as an auxiliary means of measuring traditional fish diversity.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46596151","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}
Francesco Martoni, Reannon L. Smith, Alexander M. Piper, N. Nancarrow, M. Aftab, P. Trębicki, R. Kimber, B. Rodoni, M. Blacket
Surveillance and long-term monitoring of insect pest populations are of paramount importance to limit dispersal and inform pest management. Molecular methods have been employed in diagnostics, surveillance and monitoring for the past few decades, often paired with more traditional techniques relying on morphological examinations. Within this context, the ‘iMapPESTS: Sentinel Surveillance for Agriculture’ project was conceptualised to enhance on-farm pest management decision-making via development and deployment of smart traps, able to collect insects, as well as recording associated environmental data. Here, we compared an iMapPESTS ‘Sentinel’ smart trap to an alternative suction trap over a 10-week period. We used a non-destructive insect metabarcoding approach complemented by insect morphological diagnostics to assess and compare aphid species presence and diversity across trap samples and time. Furthermore, we paired this with environmental data recorded throughout the sampling period. This methodology recorded a total of 497 different taxa from 70 traps over a 10-week period in the grain-growing region in western Victoria. This included not only the 14 aphid target species, but an additional 12 aphid species, including a new record for Victoria. Ultimately, with more than 450 bycatch species detected, this highlighted the value of insect metabarcoding, not only for pest surveillance, but also at a broader ecosystem level, with potential applications in integrated pest management and biocontrol.
{"title":"Non-destructive insect metabarcoding as a surveillance tool for the Australian grains industry: a first trial for the iMapPESTS smart trap","authors":"Francesco Martoni, Reannon L. Smith, Alexander M. Piper, N. Nancarrow, M. Aftab, P. Trębicki, R. Kimber, B. Rodoni, M. Blacket","doi":"10.3897/mbmg.7.95650","DOIUrl":"https://doi.org/10.3897/mbmg.7.95650","url":null,"abstract":"Surveillance and long-term monitoring of insect pest populations are of paramount importance to limit dispersal and inform pest management. Molecular methods have been employed in diagnostics, surveillance and monitoring for the past few decades, often paired with more traditional techniques relying on morphological examinations. Within this context, the ‘iMapPESTS: Sentinel Surveillance for Agriculture’ project was conceptualised to enhance on-farm pest management decision-making via development and deployment of smart traps, able to collect insects, as well as recording associated environmental data. Here, we compared an iMapPESTS ‘Sentinel’ smart trap to an alternative suction trap over a 10-week period. We used a non-destructive insect metabarcoding approach complemented by insect morphological diagnostics to assess and compare aphid species presence and diversity across trap samples and time. Furthermore, we paired this with environmental data recorded throughout the sampling period. This methodology recorded a total of 497 different taxa from 70 traps over a 10-week period in the grain-growing region in western Victoria. This included not only the 14 aphid target species, but an additional 12 aphid species, including a new record for Victoria. Ultimately, with more than 450 bycatch species detected, this highlighted the value of insect metabarcoding, not only for pest surveillance, but also at a broader ecosystem level, with potential applications in integrated pest management and biocontrol.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-02-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44336499","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}
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