Metabarcoding free-living marine nematodes using curated 18S and CO1 reference sequence databases for species-level taxonomic assignments.
Identifieur interne : 000277 ( PubMed/Corpus ); précédent : 000276; suivant : 000278Metabarcoding free-living marine nematodes using curated 18S and CO1 reference sequence databases for species-level taxonomic assignments.
Auteurs : Lara Macheriotou ; Katja Guilini ; Tania Nara Bezerra ; Bjorn Tytgat ; Dinh Tu Nguyen ; Thi Xuan Phuong Nguyen ; Febe Noppe ; Maickel Armenteros ; Fehmi Boufahja ; Annelien Rigaux ; Ann Vanreusel ; Sofie DeryckeSource :
- Ecology and evolution [ 2045-7758 ] ; 2019.
Abstract
High-throughput sequencing has the potential to describe biological communities with high efficiency yet comprehensive assessment of diversity with species-level resolution remains one of the most challenging aspects of metabarcoding studies. We investigated the utility of curated ribosomal and mitochondrial nematode reference sequence databases for determining phylum-specific species-level clustering thresholds. We compiled 438 ribosomal and 290 mitochondrial sequences which identified 99% and 94% as the species delineation clustering threshold, respectively. These thresholds were evaluated in HTS data from mock communities containing 39 nematode species as well as environmental samples from Vietnam. We compared the taxonomic description of the mocks generated by two read-merging and two clustering algorithms and the cluster-free Dada2 pipeline. Taxonomic assignment with the RDP classifier was assessed under different training sets. Our results showed that 36/39 mock nematode species were identified across the molecular markers (18S: 32, JB2: 19, JB3: 21) in UClust_ref OTUs at their respective clustering thresholds, outperforming UParse_denovo and the commonly used 97% similarity. Dada2 generated the most realistic number of ASVs (18S: 83, JB2: 75, JB3: 82), collectively identifying 30/39 mock species. The ribosomal marker outperformed the mitochondrial markers in terms of species and genus-level detections for both OTUs and ASVs. The number of taxonomic assignments of OTUs/ASVs was highest when the smallest reference database containing only nematode sequences was used and when sequences were truncated to the respective amplicon length. Overall, OTUs generated more species-level detections, which were, however, associated with higher error rates compared to ASVs. Genus-level assignments using ASVs exhibited higher accuracy and lower error rates compared to species-level assignments, suggesting that this is the most reliable pipeline for rapid assessment of alpha diversity from environmental samples.
DOI: 10.1002/ece3.4814
PubMed: 30805154
PubMed Central: PMC6374678
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Vietnam Academy of Science and Technology Hanoi Vietnam.</nlm:affiliation></affiliation></author><author><name sortKey="Noppe, Febe" sort="Noppe, Febe" uniqKey="Noppe F" first="Febe" last="Noppe">Febe Noppe</name><affiliation><nlm:affiliation>Marine Biology Research Group, Department of Biology Ghent University Ghent Belgium.</nlm:affiliation></affiliation></author><author><name sortKey="Armenteros, Maickel" sort="Armenteros, Maickel" uniqKey="Armenteros M" first="Maickel" last="Armenteros">Maickel Armenteros</name><affiliation><nlm:affiliation>Centro de Investigaciones Marinas Universidad de La Habana Habana Cuba.</nlm:affiliation></affiliation></author><author><name sortKey="Boufahja, Fehmi" sort="Boufahja, Fehmi" uniqKey="Boufahja F" first="Fehmi" last="Boufahja">Fehmi Boufahja</name><affiliation><nlm:affiliation>Laboratory of Biomonitoring of the Environment (LBE), Faculty of Sciences of Bizerte University of Carthage Carthage Tunisia.</nlm:affiliation></affiliation></author><author><name sortKey="Rigaux, Annelien" sort="Rigaux, Annelien" uniqKey="Rigaux A" first="Annelien" last="Rigaux">Annelien Rigaux</name><affiliation><nlm:affiliation>Marine Biology Research Group, Department of Biology Ghent University Ghent Belgium.</nlm:affiliation></affiliation></author><author><name sortKey="Vanreusel, Ann" sort="Vanreusel, Ann" uniqKey="Vanreusel A" first="Ann" last="Vanreusel">Ann Vanreusel</name><affiliation><nlm:affiliation>Marine Biology Research Group, Department of Biology Ghent University Ghent Belgium.</nlm:affiliation></affiliation></author><author><name sortKey="Derycke, Sofie" sort="Derycke, Sofie" uniqKey="Derycke S" first="Sofie" last="Derycke">Sofie Derycke</name><affiliation><nlm:affiliation>Marine Biology Research Group, Department of Biology Ghent University Ghent Belgium.</nlm:affiliation></affiliation><affiliation><nlm:affiliation>Aquatic Environment and Quality, Institute for Agricultural and Fisheries Research (ILVO) Oostende Belgium.</nlm:affiliation></affiliation><affiliation><nlm:affiliation>OD Taxonomy and Phylogeny, Royal Belgian Institute of Natural Sciences (RBINS) Brussel Belgium.</nlm:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2019">2019</date><idno type="RBID">pubmed:30805154</idno><idno type="pmid">30805154</idno><idno type="doi">10.1002/ece3.4814</idno><idno type="pmc">PMC6374678</idno><idno type="wicri:Area/PubMed/Corpus">000277</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">000277</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Metabarcoding free-living marine nematodes using curated 18S and CO1 reference sequence databases for species-level taxonomic assignments.</title><author><name sortKey="Macheriotou, Lara" sort="Macheriotou, Lara" uniqKey="Macheriotou L" first="Lara" last="Macheriotou">Lara Macheriotou</name><affiliation><nlm:affiliation>Marine Biology Research Group, Department of Biology Ghent University Ghent Belgium.</nlm:affiliation></affiliation></author><author><name sortKey="Guilini, Katja" sort="Guilini, Katja" uniqKey="Guilini K" first="Katja" last="Guilini">Katja Guilini</name><affiliation><nlm:affiliation>Marine Biology Research Group, Department of Biology Ghent University Ghent Belgium.</nlm:affiliation></affiliation></author><author><name sortKey="Bezerra, Tania Nara" sort="Bezerra, Tania Nara" uniqKey="Bezerra T" first="Tania Nara" last="Bezerra">Tania Nara Bezerra</name><affiliation><nlm:affiliation>Marine Biology Research Group, Department of Biology Ghent University Ghent Belgium.</nlm:affiliation></affiliation></author><author><name sortKey="Tytgat, Bjorn" sort="Tytgat, Bjorn" uniqKey="Tytgat B" first="Bjorn" last="Tytgat">Bjorn Tytgat</name><affiliation><nlm:affiliation>Marine Biology Research Group, Department of Biology Ghent University Ghent Belgium.</nlm:affiliation></affiliation></author><author><name sortKey="Nguyen, Dinh Tu" sort="Nguyen, Dinh Tu" uniqKey="Nguyen D" first="Dinh Tu" last="Nguyen">Dinh Tu Nguyen</name><affiliation><nlm:affiliation>Department of Nematology, Institute of Ecology and Biological Resources Vietnam Academy of Science and Technology Hanoi Vietnam.</nlm:affiliation></affiliation></author><author><name sortKey="Phuong Nguyen, Thi Xuan" sort="Phuong Nguyen, Thi Xuan" uniqKey="Phuong Nguyen T" first="Thi Xuan" last="Phuong Nguyen">Thi Xuan Phuong Nguyen</name><affiliation><nlm:affiliation>Department of Nematology, Institute of Ecology and Biological Resources Vietnam Academy of Science and Technology Hanoi Vietnam.</nlm:affiliation></affiliation></author><author><name sortKey="Noppe, Febe" sort="Noppe, Febe" uniqKey="Noppe F" first="Febe" last="Noppe">Febe Noppe</name><affiliation><nlm:affiliation>Marine Biology Research Group, Department of Biology Ghent University Ghent Belgium.</nlm:affiliation></affiliation></author><author><name sortKey="Armenteros, Maickel" sort="Armenteros, Maickel" uniqKey="Armenteros M" first="Maickel" last="Armenteros">Maickel Armenteros</name><affiliation><nlm:affiliation>Centro de Investigaciones Marinas Universidad de La Habana Habana Cuba.</nlm:affiliation></affiliation></author><author><name sortKey="Boufahja, Fehmi" sort="Boufahja, Fehmi" uniqKey="Boufahja F" first="Fehmi" last="Boufahja">Fehmi Boufahja</name><affiliation><nlm:affiliation>Laboratory of Biomonitoring of the Environment (LBE), Faculty of Sciences of Bizerte University of Carthage Carthage Tunisia.</nlm:affiliation></affiliation></author><author><name sortKey="Rigaux, Annelien" sort="Rigaux, Annelien" uniqKey="Rigaux A" first="Annelien" last="Rigaux">Annelien Rigaux</name><affiliation><nlm:affiliation>Marine Biology Research Group, Department of Biology Ghent University Ghent Belgium.</nlm:affiliation></affiliation></author><author><name sortKey="Vanreusel, Ann" sort="Vanreusel, Ann" uniqKey="Vanreusel A" first="Ann" last="Vanreusel">Ann Vanreusel</name><affiliation><nlm:affiliation>Marine Biology Research Group, Department of Biology Ghent University Ghent Belgium.</nlm:affiliation></affiliation></author><author><name sortKey="Derycke, Sofie" sort="Derycke, Sofie" uniqKey="Derycke S" first="Sofie" last="Derycke">Sofie Derycke</name><affiliation><nlm:affiliation>Marine Biology Research Group, Department of Biology Ghent University Ghent Belgium.</nlm:affiliation></affiliation><affiliation><nlm:affiliation>Aquatic Environment and Quality, Institute for Agricultural and Fisheries Research (ILVO) Oostende Belgium.</nlm:affiliation></affiliation><affiliation><nlm:affiliation>OD Taxonomy and Phylogeny, Royal Belgian Institute of Natural Sciences (RBINS) Brussel Belgium.</nlm:affiliation></affiliation></author></analytic><series><title level="j">Ecology and evolution</title><idno type="ISSN">2045-7758</idno><imprint><date when="2019" type="published">2019</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">High-throughput sequencing has the potential to describe biological communities with high efficiency yet comprehensive assessment of diversity with species-level resolution remains one of the most challenging aspects of metabarcoding studies. We investigated the utility of curated ribosomal and mitochondrial nematode reference sequence databases for determining phylum-specific species-level clustering thresholds. We compiled 438 ribosomal and 290 mitochondrial sequences which identified 99% and 94% as the species delineation clustering threshold, respectively. These thresholds were evaluated in HTS data from mock communities containing 39 nematode species as well as environmental samples from Vietnam. We compared the taxonomic description of the mocks generated by two read-merging and two clustering algorithms and the cluster-free Dada2 pipeline. Taxonomic assignment with the RDP classifier was assessed under different training sets. Our results showed that 36/39 mock nematode species were identified across the molecular markers (18S: 32, JB2: 19, JB3: 21) in UClust_ref OTUs at their respective clustering thresholds, outperforming UParse_denovo and the commonly used 97% similarity. Dada2 generated the most realistic number of ASVs (18S: 83, JB2: 75, JB3: 82), collectively identifying 30/39 mock species. The ribosomal marker outperformed the mitochondrial markers in terms of species and genus-level detections for both OTUs and ASVs. The number of taxonomic assignments of OTUs/ASVs was highest when the smallest reference database containing only nematode sequences was used and when sequences were truncated to the respective amplicon length. Overall, OTUs generated more species-level detections, which were, however, associated with higher error rates compared to ASVs. Genus-level assignments using ASVs exhibited higher accuracy and lower error rates compared to species-level assignments, suggesting that this is the most reliable pipeline for rapid assessment of alpha diversity from environmental samples.</div></front></TEI><pubmed><MedlineCitation Status="PubMed-not-MEDLINE" Owner="NLM"><PMID Version="1">30805154</PMID><DateRevised><Year>2020</Year><Month>10</Month><Day>01</Day></DateRevised><Article PubModel="Electronic-eCollection"><Journal><ISSN IssnType="Print">2045-7758</ISSN><JournalIssue CitedMedium="Print"><Volume>9</Volume><Issue>3</Issue><PubDate><Year>2019</Year><Month>Feb</Month></PubDate></JournalIssue><Title>Ecology and evolution</Title><ISOAbbreviation>Ecol Evol</ISOAbbreviation></Journal><ArticleTitle>Metabarcoding free-living marine nematodes using curated 18S and CO1 reference sequence databases for species-level taxonomic assignments.</ArticleTitle><Pagination><MedlinePgn>1211-1226</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1002/ece3.4814</ELocationID><Abstract><AbstractText>High-throughput sequencing has the potential to describe biological communities with high efficiency yet comprehensive assessment of diversity with species-level resolution remains one of the most challenging aspects of metabarcoding studies. We investigated the utility of curated ribosomal and mitochondrial nematode reference sequence databases for determining phylum-specific species-level clustering thresholds. We compiled 438 ribosomal and 290 mitochondrial sequences which identified 99% and 94% as the species delineation clustering threshold, respectively. These thresholds were evaluated in HTS data from mock communities containing 39 nematode species as well as environmental samples from Vietnam. We compared the taxonomic description of the mocks generated by two read-merging and two clustering algorithms and the cluster-free Dada2 pipeline. Taxonomic assignment with the RDP classifier was assessed under different training sets. Our results showed that 36/39 mock nematode species were identified across the molecular markers (18S: 32, JB2: 19, JB3: 21) in UClust_ref OTUs at their respective clustering thresholds, outperforming UParse_denovo and the commonly used 97% similarity. Dada2 generated the most realistic number of ASVs (18S: 83, JB2: 75, JB3: 82), collectively identifying 30/39 mock species. The ribosomal marker outperformed the mitochondrial markers in terms of species and genus-level detections for both OTUs and ASVs. The number of taxonomic assignments of OTUs/ASVs was highest when the smallest reference database containing only nematode sequences was used and when sequences were truncated to the respective amplicon length. Overall, OTUs generated more species-level detections, which were, however, associated with higher error rates compared to ASVs. Genus-level assignments using ASVs exhibited higher accuracy and lower error rates compared to species-level assignments, suggesting that this is the most reliable pipeline for rapid assessment of alpha diversity from environmental samples.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Macheriotou</LastName><ForeName>Lara</ForeName><Initials>L</Initials><Identifier Source="ORCID">https://orcid.org/0000-0002-5662-5689</Identifier><AffiliationInfo><Affiliation>Marine Biology Research Group, Department of Biology Ghent University Ghent Belgium.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Guilini</LastName><ForeName>Katja</ForeName><Initials>K</Initials><Identifier Source="ORCID">https://orcid.org/0000-0003-1832-8875</Identifier><AffiliationInfo><Affiliation>Marine Biology Research Group, Department of Biology Ghent University Ghent Belgium.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Bezerra</LastName><ForeName>Tania Nara</ForeName><Initials>TN</Initials><Identifier Source="ORCID">https://orcid.org/0000-0002-4414-7827</Identifier><AffiliationInfo><Affiliation>Marine Biology Research Group, Department of Biology Ghent University Ghent Belgium.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Tytgat</LastName><ForeName>Bjorn</ForeName><Initials>B</Initials><Identifier Source="ORCID">https://orcid.org/0000-0002-6926-3488</Identifier><AffiliationInfo><Affiliation>Marine Biology Research Group, Department of Biology Ghent University Ghent Belgium.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Nguyen</LastName><ForeName>Dinh Tu</ForeName><Initials>DT</Initials><AffiliationInfo><Affiliation>Department of Nematology, Institute of Ecology and Biological Resources Vietnam Academy of Science and Technology Hanoi Vietnam.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Phuong Nguyen</LastName><ForeName>Thi Xuan</ForeName><Initials>TX</Initials><Identifier Source="ORCID">https://orcid.org/0000-0001-8911-014X</Identifier><AffiliationInfo><Affiliation>Department of Nematology, Institute of Ecology and Biological Resources Vietnam Academy of Science and Technology Hanoi Vietnam.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Noppe</LastName><ForeName>Febe</ForeName><Initials>F</Initials><AffiliationInfo><Affiliation>Marine Biology Research Group, Department of Biology Ghent University Ghent Belgium.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Armenteros</LastName><ForeName>Maickel</ForeName><Initials>M</Initials><Identifier Source="ORCID">https://orcid.org/0000-0002-9736-9247</Identifier><AffiliationInfo><Affiliation>Centro de Investigaciones Marinas Universidad de La Habana Habana Cuba.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Boufahja</LastName><ForeName>Fehmi</ForeName><Initials>F</Initials><AffiliationInfo><Affiliation>Laboratory of Biomonitoring of the Environment (LBE), Faculty of Sciences of Bizerte University of Carthage Carthage Tunisia.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Rigaux</LastName><ForeName>Annelien</ForeName><Initials>A</Initials><AffiliationInfo><Affiliation>Marine Biology Research Group, Department of Biology Ghent University Ghent Belgium.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Vanreusel</LastName><ForeName>Ann</ForeName><Initials>A</Initials><AffiliationInfo><Affiliation>Marine Biology Research Group, Department of Biology Ghent University Ghent Belgium.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Derycke</LastName><ForeName>Sofie</ForeName><Initials>S</Initials><AffiliationInfo><Affiliation>Marine Biology Research Group, Department of Biology Ghent University Ghent Belgium.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Aquatic Environment and Quality, Institute for Agricultural and Fisheries Research (ILVO) Oostende Belgium.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>OD Taxonomy and Phylogeny, Royal Belgian Institute of Natural Sciences (RBINS) Brussel Belgium.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2019</Year><Month>01</Month><Day>22</Day></ArticleDate></Article><MedlineJournalInfo><Country>England</Country><MedlineTA>Ecol Evol</MedlineTA><NlmUniqueID>101566408</NlmUniqueID><ISSNLinking>2045-7758</ISSNLinking></MedlineJournalInfo><KeywordList Owner="NOTNLM"><Keyword MajorTopicYN="N">Nematoda</Keyword><Keyword MajorTopicYN="N">UClust</Keyword><Keyword MajorTopicYN="N">USearch9</Keyword><Keyword 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