Be positive: customized reference databases and new, local barcodes balance false taxonomic assignments in metabarcoding studies
Background In metabarcoding analyses, the taxonomic assignment is crucial to place sequencing data in biological and ecological contexts. This fundamental step depends on a reference database, which should have a good taxonomic coverage to avoid unassigned sequences. However, this goal is rarely ach...
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| Format: | Article |
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PeerJ Inc.
2023-01-01
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| Series: | PeerJ |
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| Online Access: | https://peerj.com/articles/14616.pdf |
| _version_ | 1797420231354744832 |
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| author | Francesco Mugnai Federica Costantini Anne Chenuil Michèle Leduc José Miguel Gutiérrez Ortega Emese Meglécz |
| author_facet | Francesco Mugnai Federica Costantini Anne Chenuil Michèle Leduc José Miguel Gutiérrez Ortega Emese Meglécz |
| author_sort | Francesco Mugnai |
| collection | DOAJ |
| description | Background In metabarcoding analyses, the taxonomic assignment is crucial to place sequencing data in biological and ecological contexts. This fundamental step depends on a reference database, which should have a good taxonomic coverage to avoid unassigned sequences. However, this goal is rarely achieved in many geographic regions and for several taxonomic groups. On the other hand, more is not necessarily better, as sequences in reference databases belonging to taxonomic groups out of the studied region/environment context might lead to false assignments. Methods We investigated the effect of using several subsets of a cytochrome c oxidase subunit I (COI) reference database on taxonomic assignment. Published metabarcoding sequences from the Mediterranean Sea were assigned to taxa using COInr, which is a comprehensive, non-redundant and recent database of COI sequences obtained both from BOLD and NCBI, and two of its subsets: (i) all sequences except insects (COInr-WO-Insecta), which represent the overwhelming majority of COInr database, but are irrelevant for marine samples, and (ii) all sequences from taxonomic families present in the Mediterranean Sea (COInr-Med). Four different algorithms for taxonomic assignment were employed in parallel to evaluate differences in their output and data consistency. Results The reduction of the database to more specific custom subsets increased the number of unassigned sequences. Nevertheless, since most of them were incorrectly assigned by the less specific databases, this is a positive outcome. Moreover, the taxonomic resolution (the lowest taxonomic level to which a sequence is attributed) of several sequences tended to increase when using customized databases. These findings clearly indicated the need for customized databases adapted to each study. However, the very high proportion of unassigned sequences points to the need to enrich the local database with new barcodes specifically obtained from the studied region and/or taxonomic group. Including novel local barcodes to the COI database proved to be very profitable: by adding only 116 new barcodes sequenced in our laboratory, thus increasing the reference database by only 0.04%, we were able to improve the resolution for ca. 0.6–1% of the Amplicon Sequence Variants (ASVs). |
| first_indexed | 2024-03-09T06:58:38Z |
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| institution | Directory Open Access Journal |
| issn | 2167-8359 |
| language | English |
| last_indexed | 2024-03-09T06:58:38Z |
| publishDate | 2023-01-01 |
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| spelling | doaj.art-f3606d7d479249478dc4e0e1ec7ed7be2023-12-03T09:59:49ZengPeerJ Inc.PeerJ2167-83592023-01-0111e1461610.7717/peerj.14616Be positive: customized reference databases and new, local barcodes balance false taxonomic assignments in metabarcoding studiesFrancesco Mugnai0Federica Costantini1Anne Chenuil2Michèle Leduc3José Miguel Gutiérrez Ortega4Emese Meglécz5Department of Biological, Geological and Environmental Sciences (BiGeA), University of Bologna, Ravenna, ItalyDepartment of Biological, Geological and Environmental Sciences (BiGeA), University of Bologna, Ravenna, ItalyAix Marseille Univ, Avignon Université, CNRS, IRD, IMBE, Marseille, FranceSTARESO marine station, Calvi, Corse, FranceTAXON Estudios Ambientales S.L., Alcantarilla (Murcia), SpainAix Marseille Univ, Avignon Université, CNRS, IRD, IMBE, Marseille, FranceBackground In metabarcoding analyses, the taxonomic assignment is crucial to place sequencing data in biological and ecological contexts. This fundamental step depends on a reference database, which should have a good taxonomic coverage to avoid unassigned sequences. However, this goal is rarely achieved in many geographic regions and for several taxonomic groups. On the other hand, more is not necessarily better, as sequences in reference databases belonging to taxonomic groups out of the studied region/environment context might lead to false assignments. Methods We investigated the effect of using several subsets of a cytochrome c oxidase subunit I (COI) reference database on taxonomic assignment. Published metabarcoding sequences from the Mediterranean Sea were assigned to taxa using COInr, which is a comprehensive, non-redundant and recent database of COI sequences obtained both from BOLD and NCBI, and two of its subsets: (i) all sequences except insects (COInr-WO-Insecta), which represent the overwhelming majority of COInr database, but are irrelevant for marine samples, and (ii) all sequences from taxonomic families present in the Mediterranean Sea (COInr-Med). Four different algorithms for taxonomic assignment were employed in parallel to evaluate differences in their output and data consistency. Results The reduction of the database to more specific custom subsets increased the number of unassigned sequences. Nevertheless, since most of them were incorrectly assigned by the less specific databases, this is a positive outcome. Moreover, the taxonomic resolution (the lowest taxonomic level to which a sequence is attributed) of several sequences tended to increase when using customized databases. These findings clearly indicated the need for customized databases adapted to each study. However, the very high proportion of unassigned sequences points to the need to enrich the local database with new barcodes specifically obtained from the studied region and/or taxonomic group. Including novel local barcodes to the COI database proved to be very profitable: by adding only 116 new barcodes sequenced in our laboratory, thus increasing the reference database by only 0.04%, we were able to improve the resolution for ca. 0.6–1% of the Amplicon Sequence Variants (ASVs).https://peerj.com/articles/14616.pdfReference databasesMetabarcodingCOITaxonomic assignmentMarine taxaCOInr |
| spellingShingle | Francesco Mugnai Federica Costantini Anne Chenuil Michèle Leduc José Miguel Gutiérrez Ortega Emese Meglécz Be positive: customized reference databases and new, local barcodes balance false taxonomic assignments in metabarcoding studies PeerJ Reference databases Metabarcoding COI Taxonomic assignment Marine taxa COInr |
| title | Be positive: customized reference databases and new, local barcodes balance false taxonomic assignments in metabarcoding studies |
| title_full | Be positive: customized reference databases and new, local barcodes balance false taxonomic assignments in metabarcoding studies |
| title_fullStr | Be positive: customized reference databases and new, local barcodes balance false taxonomic assignments in metabarcoding studies |
| title_full_unstemmed | Be positive: customized reference databases and new, local barcodes balance false taxonomic assignments in metabarcoding studies |
| title_short | Be positive: customized reference databases and new, local barcodes balance false taxonomic assignments in metabarcoding studies |
| title_sort | be positive customized reference databases and new local barcodes balance false taxonomic assignments in metabarcoding studies |
| topic | Reference databases Metabarcoding COI Taxonomic assignment Marine taxa COInr |
| url | https://peerj.com/articles/14616.pdf |
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