Sources of uncertainty in DNA metabarcoding of whole communities: Implications for its use in biomonitoring
Abstract Advancements have been made in the use of DNA‐based methods for the detection of single species. However, the routine application of DNA‐based methods to monitor whole communities using a metabarcoding approach and derive ecosystem status continues to be limited. We undertook a structured e...
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2025-08-01
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| author | J. Iwan Jones Amanda Arnold Dominik Buchner John F. Murphy Jasmine N. Childress Pedro Beja Florian Altermatt Kristian Meissner Ian Adams Meri Anderson Yaisel J. Borrell Andrew G. Briscoe Kat Bruce Cátia Chaves Chris Conyers Kristy Deiner Vasco Elbrecht Álvaro Fueyo Tim Goodall Javier Granero‐Castro Cristina Hartmann‐Fatu Eleanor Jones Elvira Mächler Filipa M. S. Martins Sam McGreig Lyndall Pereira‐da‐Conceicoa James L. Pretty Daniel S. Read Ben Price Florian Leese Rosetta C. Blackman |
| author_facet | J. Iwan Jones Amanda Arnold Dominik Buchner John F. Murphy Jasmine N. Childress Pedro Beja Florian Altermatt Kristian Meissner Ian Adams Meri Anderson Yaisel J. Borrell Andrew G. Briscoe Kat Bruce Cátia Chaves Chris Conyers Kristy Deiner Vasco Elbrecht Álvaro Fueyo Tim Goodall Javier Granero‐Castro Cristina Hartmann‐Fatu Eleanor Jones Elvira Mächler Filipa M. S. Martins Sam McGreig Lyndall Pereira‐da‐Conceicoa James L. Pretty Daniel S. Read Ben Price Florian Leese Rosetta C. Blackman |
| author_sort | J. Iwan Jones |
| collection | DOAJ |
| description | Abstract Advancements have been made in the use of DNA‐based methods for the detection of single species. However, the routine application of DNA‐based methods to monitor whole communities using a metabarcoding approach and derive ecosystem status continues to be limited. We undertook a structured experiment to assess factors influencing the precision and accuracy of molecular methods for freshwater macroinvertebrate community assessment. Macroinvertebrates were sorted, identified and counted from kick‐net samples using standard morphometric protocols, then reconstituted. These bulk specimen samples were then homogenised. From each bulk sample, aliquots of the homogenate were distributed among seven laboratories across Europe for DNA extraction, PCR amplification, library preparation and sequencing. Additionally, each laboratory was provided with a DNA extract from the bulk homogenate to allow for amplification through to sequencing, in order to assess the influence of DNA extraction. Hierarchical nested analyses were used to identify sources of uncertainty in the data and explore factors influencing the returned community and metrics of ecosystem quality. Furthermore, comparison with morphological analysis enabled us to benchmark molecular to traditional processing approaches. Metrics derived by morphological and DNA‐based methods differed substantially. The strongest methodological influences on variance in metrics derived from DNA‐based methods were attributed to sequencing (read count per sample) and laboratory (strong variation within samples). Furthermore, the performance of the DNA‐based method varied among samples, most likely due to differences in the composition of the invertebrate community. Logistic regression indicated that detectability did not vary among taxa, but that taxa occurring in low abundance (less than five individuals in our samples) were more likely to be missed via metabarcoding. Our findings highlight sources of uncertainty influencing DNA‐based methods and, particularly, the variability of results generated from individual laboratories. Our findings underline the need for clear guidelines, standardisation and quality assurance schemes. |
| format | Article |
| id | doaj-art-e0ee0f57f99347ef9f74062cbb6360d7 |
| institution | Kabale University |
| issn | 2041-210X |
| language | English |
| publishDate | 2025-08-01 |
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| series | Methods in Ecology and Evolution |
| spelling | doaj-art-e0ee0f57f99347ef9f74062cbb6360d72025-08-20T04:02:09ZengWileyMethods in Ecology and Evolution2041-210X2025-08-011681658167310.1111/2041-210X.70093Sources of uncertainty in DNA metabarcoding of whole communities: Implications for its use in biomonitoringJ. Iwan Jones0Amanda Arnold1Dominik Buchner2John F. Murphy3Jasmine N. Childress4Pedro Beja5Florian Altermatt6Kristian Meissner7Ian Adams8Meri Anderson9Yaisel J. Borrell10Andrew G. Briscoe11Kat Bruce12Cátia Chaves13Chris Conyers14Kristy Deiner15Vasco Elbrecht16Álvaro Fueyo17Tim Goodall18Javier Granero‐Castro19Cristina Hartmann‐Fatu20Eleanor Jones21Elvira Mächler22Filipa M. S. Martins23Sam McGreig24Lyndall Pereira‐da‐Conceicoa25James L. Pretty26Daniel S. Read27Ben Price28Florian Leese29Rosetta C. Blackman30School of Biological and Behavioural Sciences Queen Mary University of London London UKSchool of Biological and Behavioural Sciences Queen Mary University of London London UKUniversity of Duisburg‐Essen Aquatic Ecosystem Research Essen GermanySchool of Biological and Behavioural Sciences Queen Mary University of London London UKIndependent Researcher & Consultant Zürich SwitzerlandCIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, InBIO Laboratório Associado Universidade do Porto Vairão PortugalDepartment of Aquatic Ecology Eawag: Swiss Federal Institute of Aquatic Science and Technology Dübendorf SwitzerlandFinnish Environment Institute, Syke Jyväskylä FinlandFera Science York UKNatureMetrics, 1 Occam Court, Surrey Technology Centre Guildford UKFunctional Biology Department University of Oviedo Oviedo SpainNatureMetrics, 1 Occam Court, Surrey Technology Centre Guildford UKNatureMetrics, 1 Occam Court, Surrey Technology Centre Guildford UKCIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, InBIO Laboratório Associado Universidade do Porto Vairão PortugalFera Science York UKIndependent Researcher & Consultant Zürich SwitzerlandIndependent Researcher & Consultant Zürich SwitzerlandFunctional Biology Department University of Oviedo Oviedo SpainUK Centre of Ecology & Hydrology Wallingford UKEnvironment and Sustainability Area Taxus Medio Ambiente Oviedo SpainUniversity of Duisburg‐Essen Aquatic Ecosystem Research Essen GermanyFera Science York UKSimplexDNA Winterthur SwitzerlandCIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, InBIO Laboratório Associado Universidade do Porto Vairão PortugalFera Science York UKDepartment of Natural Sciences National Museums Scotland Edinburgh UKSchool of Biological and Behavioural Sciences Queen Mary University of London London UKUK Centre of Ecology & Hydrology Wallingford UKNatural History Museum London UKUniversity of Duisburg‐Essen Aquatic Ecosystem Research Essen GermanyDepartment of Aquatic Ecology Eawag: Swiss Federal Institute of Aquatic Science and Technology Dübendorf SwitzerlandAbstract Advancements have been made in the use of DNA‐based methods for the detection of single species. However, the routine application of DNA‐based methods to monitor whole communities using a metabarcoding approach and derive ecosystem status continues to be limited. We undertook a structured experiment to assess factors influencing the precision and accuracy of molecular methods for freshwater macroinvertebrate community assessment. Macroinvertebrates were sorted, identified and counted from kick‐net samples using standard morphometric protocols, then reconstituted. These bulk specimen samples were then homogenised. From each bulk sample, aliquots of the homogenate were distributed among seven laboratories across Europe for DNA extraction, PCR amplification, library preparation and sequencing. Additionally, each laboratory was provided with a DNA extract from the bulk homogenate to allow for amplification through to sequencing, in order to assess the influence of DNA extraction. Hierarchical nested analyses were used to identify sources of uncertainty in the data and explore factors influencing the returned community and metrics of ecosystem quality. Furthermore, comparison with morphological analysis enabled us to benchmark molecular to traditional processing approaches. Metrics derived by morphological and DNA‐based methods differed substantially. The strongest methodological influences on variance in metrics derived from DNA‐based methods were attributed to sequencing (read count per sample) and laboratory (strong variation within samples). Furthermore, the performance of the DNA‐based method varied among samples, most likely due to differences in the composition of the invertebrate community. Logistic regression indicated that detectability did not vary among taxa, but that taxa occurring in low abundance (less than five individuals in our samples) were more likely to be missed via metabarcoding. Our findings highlight sources of uncertainty influencing DNA‐based methods and, particularly, the variability of results generated from individual laboratories. Our findings underline the need for clear guidelines, standardisation and quality assurance schemes.https://doi.org/10.1111/2041-210X.70093macroinvertebratereproducabilityspecies detection Water Framework Directive |
| spellingShingle | J. Iwan Jones Amanda Arnold Dominik Buchner John F. Murphy Jasmine N. Childress Pedro Beja Florian Altermatt Kristian Meissner Ian Adams Meri Anderson Yaisel J. Borrell Andrew G. Briscoe Kat Bruce Cátia Chaves Chris Conyers Kristy Deiner Vasco Elbrecht Álvaro Fueyo Tim Goodall Javier Granero‐Castro Cristina Hartmann‐Fatu Eleanor Jones Elvira Mächler Filipa M. S. Martins Sam McGreig Lyndall Pereira‐da‐Conceicoa James L. Pretty Daniel S. Read Ben Price Florian Leese Rosetta C. Blackman Sources of uncertainty in DNA metabarcoding of whole communities: Implications for its use in biomonitoring Methods in Ecology and Evolution macroinvertebrate reproducability species detection Water Framework Directive |
| title | Sources of uncertainty in DNA metabarcoding of whole communities: Implications for its use in biomonitoring |
| title_full | Sources of uncertainty in DNA metabarcoding of whole communities: Implications for its use in biomonitoring |
| title_fullStr | Sources of uncertainty in DNA metabarcoding of whole communities: Implications for its use in biomonitoring |
| title_full_unstemmed | Sources of uncertainty in DNA metabarcoding of whole communities: Implications for its use in biomonitoring |
| title_short | Sources of uncertainty in DNA metabarcoding of whole communities: Implications for its use in biomonitoring |
| title_sort | sources of uncertainty in dna metabarcoding of whole communities implications for its use in biomonitoring |
| topic | macroinvertebrate reproducability species detection Water Framework Directive |
| url | https://doi.org/10.1111/2041-210X.70093 |
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