Sources of uncertainty in DNA metabarcoding of whole communities: implications for its use in biomonitoring
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...
| Autores: | , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , |
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| Formato: | artículo |
| Fecha de publicación: | 2025 |
| País: | España |
| Recursos: | Universidad de Oviedo (UNIOVI) |
| Repositorio: | RUO. Repositorio Institucional de la Universidad de Oviedo |
| Idioma: | inglés |
| OAI Identifier: | oai:digibuo.uniovi.es:10651/81726 |
| Acesso em linha: | https://hdl.handle.net/10651/81726 https://dx.doi.org/10.1111/2041-210X.70093 |
| Access Level: | acceso abierto |
| Palavra-chave: | Sources of uncertainty in DNA metabarcoding of whole communities: Implications for its use in biomonitoring |
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Sources of uncertainty in DNA metabarcoding of whole communities: implications for its use in biomonitoringJones, J. IwanArnold, AmandaBuchner, DominikMurphy, John F.Childress, Jasmine N.Beja, PedroAltermatt, FlorianMeissner, KristianAdams, IanAnderson, MeriBorrell Pichs, Yaisel Juan|||0000-0003-2840-4384Briscoe, Andrew G.Bruce, KatChaves, CátiaConyers, ChrisDeiner, KristyElbrecht, VascoFueyo, ÁlvaroGoodall, TimGranero-Castro, JavierHartmann-Fatu, CristinaJones, EleanorMächler, ElviraMartins, Filipa M. S.McGreig, SamPereira-da-Conceicoa, LyndallPretty, James L.Read, Daniel S.Price, BenLeese, FlorianBlackman, Rosetta C.Sources of uncertainty in DNA metabarcoding of whole communities: Implications for its use in biomonitoringAdvancements 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.This paper is a result of the COST Action CA15219 'Developing new genetic tools for bioassessment of aquatic ecosystems in Europe' (DNAqua-Net), funded by the European Union. KD and VE were supported by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (Grant Agreement No. 852621).20252025-07-02journal articlehttp://purl.org/coar/resource_type/c_6501VoRhttp://purl.org/coar/version/c_970fb48d4fbd8a85info:eu-repo/semantics/articlehttps://hdl.handle.net/10651/81726https://dx.doi.org/10.1111/2041-210X.70093reponame:RUO. Repositorio Institucional de la Universidad de Oviedoinstname:Universidad de Oviedo (UNIOVI)InglésengEuropean Commission http://dx.doi.org/10.13039/501100000780 Horizon 2020 Framework Programme 852621open accesshttp://purl.org/coar/access_right/c_abf2Attribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccessoai:digibuo.uniovi.es:10651/817262026-06-07T06:38:51Z |
| dc.title.none.fl_str_mv |
Sources of uncertainty in DNA metabarcoding of whole communities: implications for its use in biomonitoring |
| title |
Sources of uncertainty in DNA metabarcoding of whole communities: implications for its use in biomonitoring |
| spellingShingle |
Sources of uncertainty in DNA metabarcoding of whole communities: implications for its use in biomonitoring Jones, J. Iwan 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_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_sort |
Sources of uncertainty in DNA metabarcoding of whole communities: implications for its use in biomonitoring |
| dc.creator.none.fl_str_mv |
Jones, J. Iwan Arnold, Amanda Buchner, Dominik Murphy, John F. Childress, Jasmine N. Beja, Pedro Altermatt, Florian Meissner, Kristian Adams, Ian Anderson, Meri Borrell Pichs, Yaisel Juan|||0000-0003-2840-4384 Briscoe, Andrew G. Bruce, Kat Chaves, Cátia Conyers, Chris Deiner, Kristy Elbrecht, Vasco Fueyo, Álvaro Goodall, Tim Granero-Castro, Javier Hartmann-Fatu, Cristina Jones, Eleanor Mächler, Elvira Martins, Filipa M. S. McGreig, Sam Pereira-da-Conceicoa, Lyndall Pretty, James L. Read, Daniel S. Price, Ben Leese, Florian Blackman, Rosetta C. |
| author |
Jones, J. Iwan |
| author_facet |
Jones, J. Iwan Arnold, Amanda Buchner, Dominik Murphy, John F. Childress, Jasmine N. Beja, Pedro Altermatt, Florian Meissner, Kristian Adams, Ian Anderson, Meri Borrell Pichs, Yaisel Juan|||0000-0003-2840-4384 Briscoe, Andrew G. Bruce, Kat Chaves, Cátia Conyers, Chris Deiner, Kristy Elbrecht, Vasco Fueyo, Álvaro Goodall, Tim Granero-Castro, Javier Hartmann-Fatu, Cristina Jones, Eleanor Mächler, Elvira Martins, Filipa M. S. McGreig, Sam Pereira-da-Conceicoa, Lyndall Pretty, James L. Read, Daniel S. Price, Ben Leese, Florian Blackman, Rosetta C. |
| author_role |
author |
| author2 |
Arnold, Amanda Buchner, Dominik Murphy, John F. Childress, Jasmine N. Beja, Pedro Altermatt, Florian Meissner, Kristian Adams, Ian Anderson, Meri Borrell Pichs, Yaisel Juan|||0000-0003-2840-4384 Briscoe, Andrew G. Bruce, Kat Chaves, Cátia Conyers, Chris Deiner, Kristy Elbrecht, Vasco Fueyo, Álvaro Goodall, Tim Granero-Castro, Javier Hartmann-Fatu, Cristina Jones, Eleanor Mächler, Elvira Martins, Filipa M. S. McGreig, Sam Pereira-da-Conceicoa, Lyndall Pretty, James L. Read, Daniel S. Price, Ben Leese, Florian Blackman, Rosetta C. |
| author2_role |
author author author author author author author author author author author author author author author author author author author author author author author author author author author author author author |
| dc.subject.none.fl_str_mv |
Sources of uncertainty in DNA metabarcoding of whole communities: Implications for its use in biomonitoring |
| topic |
Sources of uncertainty in DNA metabarcoding of whole communities: Implications for its use in biomonitoring |
| description |
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. |
| publishDate |
2025 |
| dc.date.none.fl_str_mv |
2025 2025-07-02 |
| dc.type.none.fl_str_mv |
journal article http://purl.org/coar/resource_type/c_6501 VoR http://purl.org/coar/version/c_970fb48d4fbd8a85 |
| dc.type.openaire.fl_str_mv |
info:eu-repo/semantics/article |
| format |
article |
| dc.identifier.none.fl_str_mv |
https://hdl.handle.net/10651/81726 https://dx.doi.org/10.1111/2041-210X.70093 |
| url |
https://hdl.handle.net/10651/81726 https://dx.doi.org/10.1111/2041-210X.70093 |
| dc.language.none.fl_str_mv |
Inglés eng |
| language_invalid_str_mv |
Inglés |
| language |
eng |
| dc.relation.none.fl_str_mv |
European Commission http://dx.doi.org/10.13039/501100000780 Horizon 2020 Framework Programme 852621 |
| dc.rights.none.fl_str_mv |
open access http://purl.org/coar/access_right/c_abf2 Attribution 4.0 International http://creativecommons.org/licenses/by/4.0/ |
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info:eu-repo/semantics/openAccess |
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open access http://purl.org/coar/access_right/c_abf2 Attribution 4.0 International http://creativecommons.org/licenses/by/4.0/ |
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openAccess |
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reponame:RUO. Repositorio Institucional de la Universidad de Oviedo instname:Universidad de Oviedo (UNIOVI) |
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Universidad de Oviedo (UNIOVI) |
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