Eugenia Naro‐Maciel, Melissa R. Ingala, I. Werner, Brendan N. Reid, Allison M. Fitzgerald
In this data paper, we describe environmental DNA (eDNA) cytochrome c oxidase (COI) amplicon sequence data from New York City’s Bronx River Estuary. As urban systems continue to expand, describing and monitoring their biodiversity is increasingly important for sustainability. Once polluted and overexploited, New York City’s Bronx River Estuary is undergoing revitalization and restoration. To investigate and characterize the area’s diversity, we collected and sequenced river sediment and surface water samples from Hunts Point Riverside and Soundview Parks (ntotal = 48; nsediment = 25; nwater = 23). COI analysis using universal primers mlCOIintF and jgHCO2198 detected 27,328 Amplicon Sequence Variants (ASVs) from 7,653,541 sequences, and rarefaction curves reached asymptotes indicating sufficient sampling depth. Of these, eukaryotes represented 9,841ASVs from 3,562,254 sequences. At the study sites over the sampling period, community composition varied by substrate (river sediment versus surface water) and with water temperature, but not pH. The three most common phyla were Bacillariophyta (diatoms), Annelida (segmented worms), and Ochrophyta (e.g. brown and golden algae). Of the eukaryotic ASVs, we identified 614 (6.2%) to species level, including several dinoflagellates linked to Harmful Algal Blooms such as Heterocapsa spp., as well as the invasive amphipod Grandidierella japonica. The analysis detected common bivalves including blue (Mytilus edulis) and ribbed (Geukensia demissa) mussels, as well as soft-shell clams (Mya arenaria), in addition to Eastern oysters (Crassostrea virginica) that are being reintroduced to the area. Fish species undergoing restoration such as river herring (Alosa pseudoharengus, A. aestivalis) failed to be identified, although relatively common fish including Atlantic silversides (Menidia menidia), menhaden (Brevoortia tyrannus), striped bass (Morone saxatilis), and mummichogs (Fundulus heteroclitus) were found. The data highlight the utility of eDNA metabarcoding for analyzing urban estuarine biodiversity and provide a baseline for future work in the area.
{"title":"COI amplicon sequence data of environmental DNA collected from the Bronx River Estuary, New York City","authors":"Eugenia Naro‐Maciel, Melissa R. Ingala, I. Werner, Brendan N. Reid, Allison M. Fitzgerald","doi":"10.3897/mbmg.6.80139","DOIUrl":"https://doi.org/10.3897/mbmg.6.80139","url":null,"abstract":"In this data paper, we describe environmental DNA (eDNA) cytochrome c oxidase (COI) amplicon sequence data from New York City’s Bronx River Estuary. As urban systems continue to expand, describing and monitoring their biodiversity is increasingly important for sustainability. Once polluted and overexploited, New York City’s Bronx River Estuary is undergoing revitalization and restoration. To investigate and characterize the area’s diversity, we collected and sequenced river sediment and surface water samples from Hunts Point Riverside and Soundview Parks (ntotal = 48; nsediment = 25; nwater = 23). COI analysis using universal primers mlCOIintF and jgHCO2198 detected 27,328 Amplicon Sequence Variants (ASVs) from 7,653,541 sequences, and rarefaction curves reached asymptotes indicating sufficient sampling depth. Of these, eukaryotes represented 9,841ASVs from 3,562,254 sequences. At the study sites over the sampling period, community composition varied by substrate (river sediment versus surface water) and with water temperature, but not pH. The three most common phyla were Bacillariophyta (diatoms), Annelida (segmented worms), and Ochrophyta (e.g. brown and golden algae). Of the eukaryotic ASVs, we identified 614 (6.2%) to species level, including several dinoflagellates linked to Harmful Algal Blooms such as Heterocapsa spp., as well as the invasive amphipod Grandidierella japonica. The analysis detected common bivalves including blue (Mytilus edulis) and ribbed (Geukensia demissa) mussels, as well as soft-shell clams (Mya arenaria), in addition to Eastern oysters (Crassostrea virginica) that are being reintroduced to the area. Fish species undergoing restoration such as river herring (Alosa pseudoharengus, A. aestivalis) failed to be identified, although relatively common fish including Atlantic silversides (Menidia menidia), menhaden (Brevoortia tyrannus), striped bass (Morone saxatilis), and mummichogs (Fundulus heteroclitus) were found. The data highlight the utility of eDNA metabarcoding for analyzing urban estuarine biodiversity and provide a baseline for future work in the area.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-06-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45978579","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}
To test the feasibility of a citizen science program for fish eDNA metabarcoding in coastal marine environments, we recruited six groups of voluntary citizens for a science education course at a natural history museum. We held a seminar on eDNA and a workshop for seawater sampling and on-site filtration using syringes and filter cartridges for the participants. After that, they selected single survey sites following the guidelines for conducting a safe field trip. They performed seawater sampling and on-site filtration at these sites during their summer holidays. The six selected sites unexpectedly included diverse coastal habitats within a 40 km radius, located at temperate latitudes in central Japan (~35°N). After the field trips, they returned filtered cartridges to the museum, and we extracted eDNA from the filters. We performed fish eDNA metabarcoding, along with data analysis. Consequently, we identified 140 fish species across 66 families and 118 genera from the six samples, with species richness ranging from 14 to 66. Despite its limited sample size, such a diverse taxonomic range of fish species exhibited spatial biodiversity patterns within the region, which are consistent with species distribution. These include north-south and urbanization gradients of species richness, geographic structure of the fish communities, and varying salinity preferences of the component species. This case study demonstrates the potential of fish eDNA metabarcoding as an educational and scientific tool to raise public awareness and perform large-scale citizen science initiatives encompassing regional, national, or global fauna.
{"title":"The use of citizen science in fish eDNA metabarcoding for evaluating regional biodiversity in a coastal marine region: A pilot study","authors":"M. Miya, T. Sado, S. Oka, Takehiko Fukuchi","doi":"10.3897/mbmg.6.80444","DOIUrl":"https://doi.org/10.3897/mbmg.6.80444","url":null,"abstract":"To test the feasibility of a citizen science program for fish eDNA metabarcoding in coastal marine environments, we recruited six groups of voluntary citizens for a science education course at a natural history museum. We held a seminar on eDNA and a workshop for seawater sampling and on-site filtration using syringes and filter cartridges for the participants. After that, they selected single survey sites following the guidelines for conducting a safe field trip. They performed seawater sampling and on-site filtration at these sites during their summer holidays. The six selected sites unexpectedly included diverse coastal habitats within a 40 km radius, located at temperate latitudes in central Japan (~35°N). After the field trips, they returned filtered cartridges to the museum, and we extracted eDNA from the filters. We performed fish eDNA metabarcoding, along with data analysis. Consequently, we identified 140 fish species across 66 families and 118 genera from the six samples, with species richness ranging from 14 to 66. Despite its limited sample size, such a diverse taxonomic range of fish species exhibited spatial biodiversity patterns within the region, which are consistent with species distribution. These include north-south and urbanization gradients of species richness, geographic structure of the fish communities, and varying salinity preferences of the component species. This case study demonstrates the potential of fish eDNA metabarcoding as an educational and scientific tool to raise public awareness and perform large-scale citizen science initiatives encompassing regional, national, or global fauna.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44172798","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}
F. Keck, Samuel Hürlemann, Nadine Locher, C. Stamm, Kristy Deiner, F. Altermatt
Monitoring biodiversity is essential to understand the impacts of human activities and for effective management of ecosystems. Thereby, biodiversity can be assessed through direct collection of targeted organisms, through indirect evidence of their presence (e.g. signs, environmental DNA, camera trap, etc.), or through extrapolations from species distribution and species richness models. Differences in approaches used in biodiversity assessment, however, may come with individual challenges and hinder cross-study comparability. In the context of rapidly developing techniques, we compared three different approaches in order to better understand assessments of aquatic macroinvertebrate diversity. Specifically, we compared the community composition and species richness of three orders of aquatic macroinvertebrates (mayflies, stoneflies, and caddisflies, hereafter EPT) obtained via eDNA metabarcoding and via traditional in situ kicknet sampling to catchment-level based predictions of a species richness model. We used kicknet data from 24 sites in Switzerland and compared taxonomic lists to those obtained using eDNA amplified with two different primer sets. Richness detected by these methods was compared to the independent predictions made by a statistical species richness model, that is, a generalized linear model using landscape-level features to estimate EPT diversity. Despite the ability of eDNA to consistently detect some EPT species found by traditional sampling, we found important discrepancies in community composition between the kicknet and eDNA approaches, particularly at a local scale. We found the EPT-specific primer set fwhF2/EPTDr2n, detected a greater number of targeted EPT species compared to the more general primer set mlCOIintF/HCO2198. Moreover, we found that the species richness measured by eDNA from either primer set was poorly correlated to the richness measured by kicknet sampling (Pearson correlation = 0.27) and that the richness estimated by eDNA and kicknet were poorly correlated with the prediction of the species richness model (Pearson correlation = 0.30 and 0.44, respectively). The weak relationships between the traditional kicknet sampling and eDNA with this model indicates inherent limitations in upscaling species richness estimates, and possibly a limited ability of the model to meet real world expectations. It is also possible that the number of replicates was not sufficient to detect ambiguous correlations. Future challenges include improving the accuracy and sensitivity of each approach individually, yet also acknowledging their respective limitations, in order to best meet stakeholder demands and address the biodiversity crisis we are facing.
{"title":"A triad of kicknet sampling, eDNA metabarcoding, and predictive modeling to assess richness of mayflies, stoneflies and caddisflies in rivers","authors":"F. Keck, Samuel Hürlemann, Nadine Locher, C. Stamm, Kristy Deiner, F. Altermatt","doi":"10.3897/mbmg.6.79351","DOIUrl":"https://doi.org/10.3897/mbmg.6.79351","url":null,"abstract":"Monitoring biodiversity is essential to understand the impacts of human activities and for effective management of ecosystems. Thereby, biodiversity can be assessed through direct collection of targeted organisms, through indirect evidence of their presence (e.g. signs, environmental DNA, camera trap, etc.), or through extrapolations from species distribution and species richness models. Differences in approaches used in biodiversity assessment, however, may come with individual challenges and hinder cross-study comparability. In the context of rapidly developing techniques, we compared three different approaches in order to better understand assessments of aquatic macroinvertebrate diversity. Specifically, we compared the community composition and species richness of three orders of aquatic macroinvertebrates (mayflies, stoneflies, and caddisflies, hereafter EPT) obtained via eDNA metabarcoding and via traditional in situ kicknet sampling to catchment-level based predictions of a species richness model. We used kicknet data from 24 sites in Switzerland and compared taxonomic lists to those obtained using eDNA amplified with two different primer sets. Richness detected by these methods was compared to the independent predictions made by a statistical species richness model, that is, a generalized linear model using landscape-level features to estimate EPT diversity. Despite the ability of eDNA to consistently detect some EPT species found by traditional sampling, we found important discrepancies in community composition between the kicknet and eDNA approaches, particularly at a local scale. We found the EPT-specific primer set fwhF2/EPTDr2n, detected a greater number of targeted EPT species compared to the more general primer set mlCOIintF/HCO2198. Moreover, we found that the species richness measured by eDNA from either primer set was poorly correlated to the richness measured by kicknet sampling (Pearson correlation = 0.27) and that the richness estimated by eDNA and kicknet were poorly correlated with the prediction of the species richness model (Pearson correlation = 0.30 and 0.44, respectively). The weak relationships between the traditional kicknet sampling and eDNA with this model indicates inherent limitations in upscaling species richness estimates, and possibly a limited ability of the model to meet real world expectations. It is also possible that the number of replicates was not sufficient to detect ambiguous correlations. Future challenges include improving the accuracy and sensitivity of each approach individually, yet also acknowledging their respective limitations, in order to best meet stakeholder demands and address the biodiversity crisis we are facing.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-05-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46330002","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}
L. Biessy, J. Pearman, S. Waters, M. Vandergoes, S. Wood
Molecular-based techniques offer considerable potential to provide new insights into the impact of anthropogenic stressors on lake ecosystems. Microbial communities are involved in many geochemical cycling processes in lakes and a greater understanding of their functions could assist in guiding more targeted remedial actions. Recent advances in metagenomics now make it possible to determine the functional potential of entire microbial communities. The present study investigated microbial communities and their functional potential in surface sediments collected from three lakes with differing trophic states and characteristics. Surface sediments were analysed for their nutrient and elemental contents and metagenomics and metabarcoding analysis undertaken. The nutrients content of the surface sediments did not show as distinct a gradient as water chemistry monitoring data, likely reflecting effects of other lake characteristics, in particular, depth. Metabarcoding and metagenomics revealed differing bacterial community composition and functional potential amongst lakes. Amongst the differentially abundant metabolic pathways, the most prominent were clusters in the energy and xenobiotics pathways. Differences in the energy metabolism paths of photosynthesis and oxidative phosphorylation were observed. These were most likely related to changes in the community composition and especially the presence of cyanobacteria in two of the three lakes. Xenobiotic pathways, such as those involving polycyclic aromatic hydrocarbons, were highest in the lakes with the greatest agricultural land-use in their catchment. These results highlight how microbial metagenomics can be used to gain insights into the causes of differences in trophic status amongst lakes.
{"title":"Metagenomic insights to the functional potential of sediment microbial communities in freshwater lakes","authors":"L. Biessy, J. Pearman, S. Waters, M. Vandergoes, S. Wood","doi":"10.3897/mbmg.6.79265","DOIUrl":"https://doi.org/10.3897/mbmg.6.79265","url":null,"abstract":"Molecular-based techniques offer considerable potential to provide new insights into the impact of anthropogenic stressors on lake ecosystems. Microbial communities are involved in many geochemical cycling processes in lakes and a greater understanding of their functions could assist in guiding more targeted remedial actions. Recent advances in metagenomics now make it possible to determine the functional potential of entire microbial communities. The present study investigated microbial communities and their functional potential in surface sediments collected from three lakes with differing trophic states and characteristics. Surface sediments were analysed for their nutrient and elemental contents and metagenomics and metabarcoding analysis undertaken. The nutrients content of the surface sediments did not show as distinct a gradient as water chemistry monitoring data, likely reflecting effects of other lake characteristics, in particular, depth. Metabarcoding and metagenomics revealed differing bacterial community composition and functional potential amongst lakes. Amongst the differentially abundant metabolic pathways, the most prominent were clusters in the energy and xenobiotics pathways. Differences in the energy metabolism paths of photosynthesis and oxidative phosphorylation were observed. These were most likely related to changes in the community composition and especially the presence of cyanobacteria in two of the three lakes. Xenobiotic pathways, such as those involving polycyclic aromatic hydrocarbons, were highest in the lakes with the greatest agricultural land-use in their catchment. These results highlight how microbial metagenomics can be used to gain insights into the causes of differences in trophic status amongst lakes.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-03-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42839644","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}
S. Sogawa, K. Tsuchiya, S. Nagai, S. Shimode, V. Kuwahara
Sagami Bay, Japan is influenced by both the warm Kuroshio Current and the cold Oyashio Current and rich nutrients are supplied from multiple river sources and the deep-sea, forming a dynamic ecosystem. The aim of the present study was to investigate eukaryotic and bacterial communities in the coastal waters of Sagami Bay, using 16S rRNA and 18S rRNA sequencing and to assess the seasonal and vertical dynamics in relation to physicochemical and biological conditions. Eukaryotic and bacterial communities showed synchronous seasonal and vertical changes along with environmental variability. Diversity of plankton community suspended in the surface was lower than those at the subsurface layers in both the eukaryotes and bacteria communities; however, community diversity showed different characteristics in the subsurface where the eukaryotic community at the deeper layer (100 m) was as low as the surface and highest in intermediate depth layers (10–50 m), while that of bacterial community was highest in the deeper layer (100 m). The annual variability of the coastal microbial communities was driven, not only by the seasonal changes of abiotic and biotic factors and short-term rapid changes by river water inflow and phytoplankton blooms, but also largely influenced by deep-seawater upwellings due to the unique seafloor topography.
{"title":"Annual dynamics of eukaryotic and bacterial communities revealed by 18S and 16S rRNA metabarcoding in the coastal ecosystem of Sagami Bay, Japan","authors":"S. Sogawa, K. Tsuchiya, S. Nagai, S. Shimode, V. Kuwahara","doi":"10.3897/mbmg.6.78181","DOIUrl":"https://doi.org/10.3897/mbmg.6.78181","url":null,"abstract":"Sagami Bay, Japan is influenced by both the warm Kuroshio Current and the cold Oyashio Current and rich nutrients are supplied from multiple river sources and the deep-sea, forming a dynamic ecosystem. The aim of the present study was to investigate eukaryotic and bacterial communities in the coastal waters of Sagami Bay, using 16S rRNA and 18S rRNA sequencing and to assess the seasonal and vertical dynamics in relation to physicochemical and biological conditions. Eukaryotic and bacterial communities showed synchronous seasonal and vertical changes along with environmental variability. Diversity of plankton community suspended in the surface was lower than those at the subsurface layers in both the eukaryotes and bacteria communities; however, community diversity showed different characteristics in the subsurface where the eukaryotic community at the deeper layer (100 m) was as low as the surface and highest in intermediate depth layers (10–50 m), while that of bacterial community was highest in the deeper layer (100 m). The annual variability of the coastal microbial communities was driven, not only by the seasonal changes of abiotic and biotic factors and short-term rapid changes by river water inflow and phytoplankton blooms, but also largely influenced by deep-seawater upwellings due to the unique seafloor topography.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-02-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42331076","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}
Masayuki K. Sakata, Mone U. Kawata, A. Kurabayashi, Takaki Kurita, Masatoshi Nakamura, Tomoyasu Shirako, R. Kakehashi, K. Nishikawa, Mohamad Yazid Hossman, T. Nishijima, Junichi Kabamoto, M. Miya, T. Minamoto
Biodiversity monitoring is important for the conservation of natural ecosystems in general, but particularly for amphibians, whose populations are pronouncedly declining. However, amphibians’ ecological traits (e.g. nocturnal or aquatic) often prevent their precise monitoring. Environmental DNA (eDNA) metabarcoding – analysis of extra-organismal DNA released into the environment – allows the easy and effective monitoring of the biodiversity of aquatic organisms. Here, we developed and tested the utility of original PCR primer sets. First, we conducted in vitro PCR amplification tests with universal primer candidates using total DNA extracted from amphibian tissues. Five primer sets successfully amplified the target DNA fragments (partial 16S rRNA gene fragments of 160–311 bp) from all 16 taxa tested (from the three living amphibian orders Anura, Caudata and Gymnophiona). Next, we investigated the taxonomic resolution retrieved using each primer set. The results revealed that the universal primer set “Amph16S” had the highest resolution amongst the tested sets. Finally, we applied Amph16S to the water samples collected in the field and evaluated its detection capability by comparing the species detected using eDNA and physical survey (capture-based sampling and visual survey) in multiple agricultural ecosystems across Japan (160 sites in 10 areas). The eDNA metabarcoding with Amph16S detected twice as many species as the physical surveys (16 vs. 8 species, respectively), indicating the effectiveness of Amph16S in biodiversity monitoring and ecological research for amphibian communities.
{"title":"Development and evaluation of PCR primers for environmental DNA (eDNA) metabarcoding of Amphibia","authors":"Masayuki K. Sakata, Mone U. Kawata, A. Kurabayashi, Takaki Kurita, Masatoshi Nakamura, Tomoyasu Shirako, R. Kakehashi, K. Nishikawa, Mohamad Yazid Hossman, T. Nishijima, Junichi Kabamoto, M. Miya, T. Minamoto","doi":"10.3897/mbmg.6.76534","DOIUrl":"https://doi.org/10.3897/mbmg.6.76534","url":null,"abstract":"Biodiversity monitoring is important for the conservation of natural ecosystems in general, but particularly for amphibians, whose populations are pronouncedly declining. However, amphibians’ ecological traits (e.g. nocturnal or aquatic) often prevent their precise monitoring. Environmental DNA (eDNA) metabarcoding – analysis of extra-organismal DNA released into the environment – allows the easy and effective monitoring of the biodiversity of aquatic organisms. Here, we developed and tested the utility of original PCR primer sets. First, we conducted in vitro PCR amplification tests with universal primer candidates using total DNA extracted from amphibian tissues. Five primer sets successfully amplified the target DNA fragments (partial 16S rRNA gene fragments of 160–311 bp) from all 16 taxa tested (from the three living amphibian orders Anura, Caudata and Gymnophiona). Next, we investigated the taxonomic resolution retrieved using each primer set. The results revealed that the universal primer set “Amph16S” had the highest resolution amongst the tested sets. Finally, we applied Amph16S to the water samples collected in the field and evaluated its detection capability by comparing the species detected using eDNA and physical survey (capture-based sampling and visual survey) in multiple agricultural ecosystems across Japan (160 sites in 10 areas). The eDNA metabarcoding with Amph16S detected twice as many species as the physical surveys (16 vs. 8 species, respectively), indicating the effectiveness of Amph16S in biodiversity monitoring and ecological research for amphibian communities.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47897464","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}
S. Nagai, Sirje Sildever, N. Nishi, Satoshi Tazawa, Leila Basti, Takanori Kobayashi, Y. Ishino
Accuracy of PCR amplification is vital for obtaining reliable amplicon-sequencing results by metabarcoding. Here, we performed a comparative analysis of error profiles in the PCR products by 14 different PCR kits using a mock eukaryotic community DNA sample mimicking metabarcoding analysis. To prepare a mock eukaryotic community from the marine environment, equal amounts of plasmid DNA from 40 microalgal species were mixed and used for amplicon-sequencing by a high-throughput sequencing approach. To compare the differences in PCR kits used for this experiment, we focused on the following seven parameters: 1) Quality, 2) Chimera, 3) Blast top hit accuracy, 4) Deletion, 5) Insertion, 6) Base substitution and 7) Amplification bias amongst species. The results showed statistically significant differences (p < 0.05) for all of the seven parameters depending on the PCR kits used. These differences may result from the different DNA polymerases included in each kit, although the result can also be influenced by PCR reaction conditions. Simultaneous analysis of several parameters suggested that kits containing KOD plus Neo (TOYOBO) and HotStart Taq DNA polymerase (BiONEER, CA, US) at the annealing temperature of 65 °C displayed better results in terms of parameters associated with chimeras, top hit similarity and deletions.
PCR扩增的准确性对于通过代谢条形码获得可靠的扩增子测序结果至关重要。在这里，我们使用模拟代谢编码分析的模拟真核生物群落DNA样本，通过14种不同的PCR试剂盒对PCR产物中的错误图谱进行了比较分析。为了从海洋环境中制备模拟真核生物群落，将来自40种微藻的等量质粒DNA混合，并通过高通量测序方法用于扩增子测序。为了比较本实验中使用的PCR试剂盒的差异，我们重点研究了以下七个参数：1）质量，2）嵌合体，3）Blast top hit准确性，4）缺失，5）插入，6）碱基取代和7）物种间的扩增偏差。结果显示，根据所使用的PCR试剂盒，所有七个参数的差异具有统计学意义（p<0.05）。这些差异可能是由每个试剂盒中包含的不同DNA聚合酶引起的，尽管结果也可能受到PCR反应条件的影响。对几个参数的同时分析表明，在65°C的退火温度下，含有KOD加Neo（TOYOBO）和HotStart-Taq DNA聚合酶（BiONEER，CA，US）的试剂盒在与嵌合体、顶部命中相似性和缺失相关的参数方面显示出更好的结果。
{"title":"Comparing PCR-generated artifacts of different polymerases for improved accuracy of DNA metabarcoding","authors":"S. Nagai, Sirje Sildever, N. Nishi, Satoshi Tazawa, Leila Basti, Takanori Kobayashi, Y. Ishino","doi":"10.3897/mbmg.6.77704","DOIUrl":"https://doi.org/10.3897/mbmg.6.77704","url":null,"abstract":"Accuracy of PCR amplification is vital for obtaining reliable amplicon-sequencing results by metabarcoding. Here, we performed a comparative analysis of error profiles in the PCR products by 14 different PCR kits using a mock eukaryotic community DNA sample mimicking metabarcoding analysis. To prepare a mock eukaryotic community from the marine environment, equal amounts of plasmid DNA from 40 microalgal species were mixed and used for amplicon-sequencing by a high-throughput sequencing approach. To compare the differences in PCR kits used for this experiment, we focused on the following seven parameters: 1) Quality, 2) Chimera, 3) Blast top hit accuracy, 4) Deletion, 5) Insertion, 6) Base substitution and 7) Amplification bias amongst species. The results showed statistically significant differences (p < 0.05) for all of the seven parameters depending on the PCR kits used. These differences may result from the different DNA polymerases included in each kit, although the result can also be influenced by PCR reaction conditions. Simultaneous analysis of several parameters suggested that kits containing KOD plus Neo (TOYOBO) and HotStart Taq DNA polymerase (BiONEER, CA, US) at the annealing temperature of 65 °C displayed better results in terms of parameters associated with chimeras, top hit similarity and deletions.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45976575","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}
K. Brasell, X. Pochon, J. Howarth, J. Pearman, A. Zaiko, Lucy Thompson, M. Vandergoes, K. Simon, S. Wood
Lake sediments hold a wealth of information from past environments that is highly valuable for paleolimnological reconstructions. These studies increasingly apply modern molecular tools targeting sedimentary DNA (sedDNA). However, sediment core sampling can be logistically difficult, making immediate subsampling for sedDNA challenging. Sediment cores are often refrigerated (4 °C) for weeks or months before subsampling. We investigated the impact of storage time on changes in DNA (purified or as cell lysate) concentrations and shifts in biological communities following storage of lake surface sediment at 4 °C for up to 24 weeks. Sediment samples (~ 0.22 g, in triplicate per time point) were spiked with purified DNA (100 or 200 ng) or lysate from a brackish water cyanobacterium that produces the cyanotoxin nodularin or non-spiked. Samples were analysed every 1–4 weeks over a 24-week period. Droplet digital PCR showed no significant decrease in the target gene (nodularin synthetase – subunit F; ndaF) over the 24-week period for samples spiked with purified DNA, while copy number decreased by more than half in cell lysate-spiked samples. There was significant change over time in bacteria and eukaryotic community composition assessed using metabarcoding. Amongst bacteria, the cyanobacterial signal became negligible after 5 weeks while Proteobacteria increased. In the eukaryotic community, Cercozoa became dominant after 6 weeks. These data demonstrate that DNA yields and community composition data shift significantly when sediments are stored chilled for more than 5 weeks. This highlights the need for rapid subsampling and appropriate storage of sediment core samples for paleogenomic studies.
{"title":"Shifts in DNA yield and biological community composition in stored sediment: implications for paleogenomic studies","authors":"K. Brasell, X. Pochon, J. Howarth, J. Pearman, A. Zaiko, Lucy Thompson, M. Vandergoes, K. Simon, S. Wood","doi":"10.3897/mbmg.6.78128","DOIUrl":"https://doi.org/10.3897/mbmg.6.78128","url":null,"abstract":"Lake sediments hold a wealth of information from past environments that is highly valuable for paleolimnological reconstructions. These studies increasingly apply modern molecular tools targeting sedimentary DNA (sedDNA). However, sediment core sampling can be logistically difficult, making immediate subsampling for sedDNA challenging. Sediment cores are often refrigerated (4 °C) for weeks or months before subsampling. We investigated the impact of storage time on changes in DNA (purified or as cell lysate) concentrations and shifts in biological communities following storage of lake surface sediment at 4 °C for up to 24 weeks. Sediment samples (~ 0.22 g, in triplicate per time point) were spiked with purified DNA (100 or 200 ng) or lysate from a brackish water cyanobacterium that produces the cyanotoxin nodularin or non-spiked. Samples were analysed every 1–4 weeks over a 24-week period. Droplet digital PCR showed no significant decrease in the target gene (nodularin synthetase – subunit F; ndaF) over the 24-week period for samples spiked with purified DNA, while copy number decreased by more than half in cell lysate-spiked samples. There was significant change over time in bacteria and eukaryotic community composition assessed using metabarcoding. Amongst bacteria, the cyanobacterial signal became negligible after 5 weeks while Proteobacteria increased. In the eukaryotic community, Cercozoa became dominant after 6 weeks. These data demonstrate that DNA yields and community composition data shift significantly when sediments are stored chilled for more than 5 weeks. This highlights the need for rapid subsampling and appropriate storage of sediment core samples for paleogenomic studies.","PeriodicalId":18374,"journal":{"name":"Metabarcoding and Metagenomics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46370737","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}
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}
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