Have the cake and eat it: optimizing nondestructive DNA metabarcoding of macroinvertebrate samples for freshwater biomonitoring

Detalhes bibliográficos
Autor(a) principal: Martins, Filipa M.S.
Data de Publicação: 2018
Outros Autores: Galhardo, Mafalda, Filipe, Ana Filipa, Teixeira, Amílcar, Pinheiro, Paulo, Paupério, Joana, Alves, Paulo C., Beja, Pedro
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
Texto Completo: http://hdl.handle.net/10198/20286
Resumo: DNA metabarcoding can contribute to improving cost‐effectiveness and accuracy of biological assessments of aquatic ecosystems, but significant optimization and standardization efforts are still required to mainstream its application into biomonitoring programmes. In assessments based on freshwater macroinvertebrates, a key challenge is that DNA is often extracted from cleaned, sorted and homogenized bulk samples, which is time‐consuming and may be incompatible with sample preservation requirements of regulatory agencies. Here, we optimize and evaluate metabarcoding procedures based on DNA recovered from 96% ethanol used to preserve field samples and thus including potential PCR inhibitors and nontarget organisms. We sampled macroinvertebrates at five sites and subsampled the preservative ethanol at 1 to 14 days thereafter. DNA was extracted using column‐based enzymatic (TISSUE) or mechanic (SOIL) protocols, or with a new magnetic‐based enzymatic protocol (BEAD), and a 313‐bp COI fragment was amplified. Metabarcoding detected at least 200 macroinvertebrate taxa, including most taxa detected through morphology and for which there was a reference barcode. Better results were obtained with BEAD than SOIL or TISSUE, and with subsamples taken 7–14 than 1–7 days after sampling, in terms of DNA concentration and integrity, taxa diversity and matching between metabarcoding and morphology. Most variation in community composition was explained by differences among sites, with small but significant contributions of subsampling day and extraction method, and negligible contributions of extraction and PCR replication. Our methods enhance reliability of preservative ethanol as a potential source of DNA for macroinvertebrate metabarcoding, with a strong potential application in freshwater biomonitoring.
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spelling Have the cake and eat it: optimizing nondestructive DNA metabarcoding of macroinvertebrate samples for freshwater biomonitoringBenthic macroinvertebratesDNA extractionDNA metabarcodingFreshwater bioassessmentPreservative ethanolWater framework directiveDNA metabarcoding can contribute to improving cost‐effectiveness and accuracy of biological assessments of aquatic ecosystems, but significant optimization and standardization efforts are still required to mainstream its application into biomonitoring programmes. In assessments based on freshwater macroinvertebrates, a key challenge is that DNA is often extracted from cleaned, sorted and homogenized bulk samples, which is time‐consuming and may be incompatible with sample preservation requirements of regulatory agencies. Here, we optimize and evaluate metabarcoding procedures based on DNA recovered from 96% ethanol used to preserve field samples and thus including potential PCR inhibitors and nontarget organisms. We sampled macroinvertebrates at five sites and subsampled the preservative ethanol at 1 to 14 days thereafter. DNA was extracted using column‐based enzymatic (TISSUE) or mechanic (SOIL) protocols, or with a new magnetic‐based enzymatic protocol (BEAD), and a 313‐bp COI fragment was amplified. Metabarcoding detected at least 200 macroinvertebrate taxa, including most taxa detected through morphology and for which there was a reference barcode. Better results were obtained with BEAD than SOIL or TISSUE, and with subsamples taken 7–14 than 1–7 days after sampling, in terms of DNA concentration and integrity, taxa diversity and matching between metabarcoding and morphology. Most variation in community composition was explained by differences among sites, with small but significant contributions of subsampling day and extraction method, and negligible contributions of extraction and PCR replication. Our methods enhance reliability of preservative ethanol as a potential source of DNA for macroinvertebrate metabarcoding, with a strong potential application in freshwater biomonitoring.This article has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 668981 (ERA Chair in Environmental metagenomics, EnvMetaGen) and was supported by EDP‐Biodiversity Chair (EDP/FCT) and the project FRESHING funded by the Portuguese Foundation for Science and Technology (FCT) and COMPETE (PTDC/ AAGMAA/2261/2014—POCI‐01‐0145‐FEDER‐356 016824). FMSM was supported by FCT PhD grant SFRH/BD/104703/2014. PB was supported by EDP‐Biodiversity Chair, MG and JP by the EnvMetaGen, and AFF by FRESHING/FCT. We thank B. Maia‐ Carvalho for his invaluable support on the development of the BEAD protocol, and M. Monaghan for suggestions on DNA quality assessment.Biblioteca Digital do IPBMartins, Filipa M.S.Galhardo, MafaldaFilipe, Ana FilipaTeixeira, AmílcarPinheiro, PauloPaupério, JoanaAlves, Paulo C.Beja, Pedro2018-01-19T10:00:00Z20192019-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/10198/20286engMartins, Filipa M.S.; Galhardo, Mafalda; Filipe, Ana F.; Teixeira, Amílcar; Pinheiro, Paulo; Paupério, Joana; Alves, Paulo C.; Beja, Pedro (2019). Have the cake and eat it: Optimizing nondestructive DNA metabarcoding of macroinvertebrate samples for freshwater biomonitoring. Molecular Ecology Resources. ISSN 1755-098X. 19, p. 863-8761755-098X10.1111/1755-0998.13012info:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-11-21T10:46:14Zoai:bibliotecadigital.ipb.pt:10198/20286Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T23:11:04.578750Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse
dc.title.none.fl_str_mv Have the cake and eat it: optimizing nondestructive DNA metabarcoding of macroinvertebrate samples for freshwater biomonitoring
title Have the cake and eat it: optimizing nondestructive DNA metabarcoding of macroinvertebrate samples for freshwater biomonitoring
spellingShingle Have the cake and eat it: optimizing nondestructive DNA metabarcoding of macroinvertebrate samples for freshwater biomonitoring
Martins, Filipa M.S.
Benthic macroinvertebrates
DNA extraction
DNA metabarcoding
Freshwater bioassessment
Preservative ethanol
Water framework directive
title_short Have the cake and eat it: optimizing nondestructive DNA metabarcoding of macroinvertebrate samples for freshwater biomonitoring
title_full Have the cake and eat it: optimizing nondestructive DNA metabarcoding of macroinvertebrate samples for freshwater biomonitoring
title_fullStr Have the cake and eat it: optimizing nondestructive DNA metabarcoding of macroinvertebrate samples for freshwater biomonitoring
title_full_unstemmed Have the cake and eat it: optimizing nondestructive DNA metabarcoding of macroinvertebrate samples for freshwater biomonitoring
title_sort Have the cake and eat it: optimizing nondestructive DNA metabarcoding of macroinvertebrate samples for freshwater biomonitoring
author Martins, Filipa M.S.
author_facet Martins, Filipa M.S.
Galhardo, Mafalda
Filipe, Ana Filipa
Teixeira, Amílcar
Pinheiro, Paulo
Paupério, Joana
Alves, Paulo C.
Beja, Pedro
author_role author
author2 Galhardo, Mafalda
Filipe, Ana Filipa
Teixeira, Amílcar
Pinheiro, Paulo
Paupério, Joana
Alves, Paulo C.
Beja, Pedro
author2_role author
author
author
author
author
author
author
dc.contributor.none.fl_str_mv Biblioteca Digital do IPB
dc.contributor.author.fl_str_mv Martins, Filipa M.S.
Galhardo, Mafalda
Filipe, Ana Filipa
Teixeira, Amílcar
Pinheiro, Paulo
Paupério, Joana
Alves, Paulo C.
Beja, Pedro
dc.subject.por.fl_str_mv Benthic macroinvertebrates
DNA extraction
DNA metabarcoding
Freshwater bioassessment
Preservative ethanol
Water framework directive
topic Benthic macroinvertebrates
DNA extraction
DNA metabarcoding
Freshwater bioassessment
Preservative ethanol
Water framework directive
description DNA metabarcoding can contribute to improving cost‐effectiveness and accuracy of biological assessments of aquatic ecosystems, but significant optimization and standardization efforts are still required to mainstream its application into biomonitoring programmes. In assessments based on freshwater macroinvertebrates, a key challenge is that DNA is often extracted from cleaned, sorted and homogenized bulk samples, which is time‐consuming and may be incompatible with sample preservation requirements of regulatory agencies. Here, we optimize and evaluate metabarcoding procedures based on DNA recovered from 96% ethanol used to preserve field samples and thus including potential PCR inhibitors and nontarget organisms. We sampled macroinvertebrates at five sites and subsampled the preservative ethanol at 1 to 14 days thereafter. DNA was extracted using column‐based enzymatic (TISSUE) or mechanic (SOIL) protocols, or with a new magnetic‐based enzymatic protocol (BEAD), and a 313‐bp COI fragment was amplified. Metabarcoding detected at least 200 macroinvertebrate taxa, including most taxa detected through morphology and for which there was a reference barcode. Better results were obtained with BEAD than SOIL or TISSUE, and with subsamples taken 7–14 than 1–7 days after sampling, in terms of DNA concentration and integrity, taxa diversity and matching between metabarcoding and morphology. Most variation in community composition was explained by differences among sites, with small but significant contributions of subsampling day and extraction method, and negligible contributions of extraction and PCR replication. Our methods enhance reliability of preservative ethanol as a potential source of DNA for macroinvertebrate metabarcoding, with a strong potential application in freshwater biomonitoring.
publishDate 2018
dc.date.none.fl_str_mv 2018-01-19T10:00:00Z
2019
2019-01-01T00:00:00Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://hdl.handle.net/10198/20286
url http://hdl.handle.net/10198/20286
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv Martins, Filipa M.S.; Galhardo, Mafalda; Filipe, Ana F.; Teixeira, Amílcar; Pinheiro, Paulo; Paupério, Joana; Alves, Paulo C.; Beja, Pedro (2019). Have the cake and eat it: Optimizing nondestructive DNA metabarcoding of macroinvertebrate samples for freshwater biomonitoring. Molecular Ecology Resources. ISSN 1755-098X. 19, p. 863-876
1755-098X
10.1111/1755-0998.13012
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.source.none.fl_str_mv reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
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