<scp>crabs</scp>—A software program to generate curated reference databases for metabarcoding sequencing data

Jeunen, Gert‐Jan; Dowle, Eddy; Edgecombe, Jonika; Ammon, Ulla von; Gemmell, Neil J.; Cross, Hugh

doi:10.1111/1755-0998.13741

Cited by 21 publications

(52 citation statements)

References 86 publications

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Section: Methodsmentioning

confidence: 99%

“…We created an annotated reference database of amplicon sequences associated with each primer set using the CRABS software and workflow (Jeunen et al., 2022). This effort involved downloading all arthropod mitochondrial sequences from GenBank and the Barcode of Life Database (BOLD) in May 2023, performing in silico PCR and pairwise global alignment analysis on them to extract amplicons, and performing the recommended cleaning steps to construct a final curated reference database (Jeunen et al., 2022). We used the ecotag function within OBITools to match to the reference database and assign taxonomy, using thresholds of ≥99% match to accept identifications at the species level, ≥98% at the genus level, ≥95% at the family, and ≥90% at the order level.…”

Section: Methodsmentioning

confidence: 99%

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Using surface environmental DNA to assess arthropod biodiversity within a forested ecosystem

Allen,

Lockwood,

Kwait

et al. 2023

Environmental DNA

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Terrestrial arthropods are a diverse taxonomic group of significant ecological and economic importance. Our ability to understand the diversity that comprises this group is hampered by the variety of sampling techniques and high level of taxonomic expertise required to identify individual species. DNA metabarcoding approaches have potential to overcome these challenges but have been mainly limited to studies where DNA is directly extracted from trapped individuals. We posit that collection of environmental DNA (eDNA) deposited on vegetation surfaces could provide an alternative method of conducting metabarcoding‐based arthropod inventories. In this study, we illustrate the promise of characterizing arthropod biodiversity based on eDNA collected from terrestrial plant surfaces. We collected 40 paired samples using two novel eDNA sampling techniques—tree bark and foliage sampling—in a New Jersey, USA, pine barrens forest. Metabarcoding using two primer sets revealed significantly higher taxonomic richness for the 16S versus COI primer set (1077 vs. 650 molecular operational taxonomic units; MOTUs), as well as higher richness and diversity in foliage versus bark samples. Accumulation curves suggest that our samples captured about half of the available MOTU‐level diversity. Matching to reference databases revealed 28 arthropod orders, 181 families, 353 genera, and 292 species. Despite having lower MOTU‐level richness, the COI primer set revealed more taxa that were identified to species (197 vs. 115) and genus (227 vs. 173) thanks to a more complete reference database. The two primer sets and sampling substrates showed distinct community compositions that differed in important ecological traits (feeding guild, body size), demonstrating the utility of a multi‐faceted sampling and analytical approach. Our study highlights the value of exploiting eDNA left on plant surfaces via metabarcoding for contributing to rapid arthropod inventories, and thus realizing a range of ecological research and management goals.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Using surface environmental DNA to assess arthropod biodiversity within a forested ecosystem

Allen,

Lockwood,

Kwait

et al. 2023

Environmental DNA

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“…Taxonomy assignments from BLAST results (blastn; megablast; nr/nt database; exclude "uncultured/environmental sample sequences") were processed by an in-house python script to determine the last common ancestor (LCA; Jeunen et al, 2020). For Sintax taxonomy assignments, custom curated reference databases were generated for each of the three assays using CRABS version 1.0.1 (Jeunen, Dowle, et al, 2022; GitHub: https://github.com/gjeun en/refer ence_datab ase_creator; Supporting Information 4 in Appendix S1). Reference databases were generated by downloading sequences from the NCBI nt and MitoFish databases.…”

Section: Bioinformatic Analysismentioning

confidence: 99%

Assessing the utility of marine filter feeders for environmental DNA (eDNA) biodiversity monitoring

Jeunen

Cane

Ferreira

et al. 2023

Molecular Ecology Resources

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Aquatic environmental DNA (eDNA) surveys are transforming how marine ecosystems are monitored. The time-consuming preprocessing step of active filtration, however, remains a bottleneck. Hence, new approaches that eliminate the need for active filtration are required. Filter-feeding invertebrates have been proven to collect eDNA, but side-by-side comparative studies to investigate the similarity between aquatic and filter-feeder eDNA signals are essential. Here, we investigated the differences among four eDNA sources (water; bivalve gill-tissue; sponges; and ethanol in which filter-feeding organisms were stored) along a vertically stratified transect in Doubtful Sound, New Zealand using three metabarcoding primer sets targeting fish and vertebrates. Combined, eDNA sources detected 59 vertebrates, while concurrent diver surveys observed eight fish species. There were no significant differences in alpha and beta diversity between water and sponge eDNA and both sources were highly correlated. Vertebrate eDNA was successfully extracted from the ethanol in which sponges were stored, although a reduced number of species were detected. Bivalve gill-tissue dissections, on the other hand, failed to reliably detect eDNA. Overall, our results show that vertebrate eDNA signals obtained from water samples and marine sponges are highly concordant. The strong similarity in eDNA signals demonstrates the potential of marine sponges as an additional tool for eDNA-based marine biodiversity surveys, by enabling the incorporation of larger sample numbers in eDNA surveys, reducing plastic waste, simplifying sample collection, and as a cost-efficient alternative. However, we note the importance to not detrimentally impact marine communities by, for example, nonlethal subsampling, specimen cloning, or using bycatch specimens.

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“…Finally, at the most local level, it is necessary to increase awareness and train bioinformaticians and end‐users on the problems specific to reference databases. For example, tools can be implemented to improve the quality control and curation workflows of reference databases such as taxci (Rulik et al, 2017), MetaCurator (Richardson et al, 2020), Anacapa (Curd et al, 2019), BCdatabaser (Keller et al, 2020), RESCRIPt (Robeson et al, 2021), DB4Q2 (Dubois et al, 2022), NEA_fish_DB (Claver et al, 2022), CRABS (Jeunen et al, 2022) and refdb (Keck & Altermatt, 2022). The technical solutions discussed in this paper should be used by scientists willing to compile their own database.…”

Section: Conclusion and Recommendationsmentioning

confidence: 99%

Navigating the seven challenges of taxonomic reference databases in metabarcoding analyses

Keck

Couton

Altermatt

2022

Molecular Ecology Resources

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Assessment of biodiversity using metabarcoding data, such as from bulk or environmental DNA sampling, is becoming increasingly relevant in ecology, biodiversity sciences and monitoring. Thereby, the taxonomic identification of species from their DNA sequences relies strongly on reference databases that link genetic sequences to taxonomic names. These databases vary in completeness and availability, depending on the taxonomic group studied and the genetic region targeted. The incompleteness of reference databases is an important argument to explain the nondetection by metabarcoding of species supposedly present. However, there exist further and generally overlooked problems with reference databases that can lead to false or inaccurate inferences of taxonomic assignment. Here, we synthesize all possible problems inherent to reference databases. In particular, we identify a complete, mutually nonexclusive list of seven classes of challenges when it comes to selecting, developing and using a reference database for taxonomic assignment. These are: (i) mislabelling, (ii) sequencing errors, (iii) sequence conflict, (iv) taxonomic conflict, (v) low taxonomic resolution, (vi) missing taxa and (vii) missing intraspecific variants. For each problem identified, we provide a description of possible consequences on the taxonomic assignment process. We illustrate the respective problem with examples taken from the literature or obtained by quantitative analyses of public databases, such as GenBank or BOLD. Finally, we discuss possible solutions to the identified problems and how to navigate them. Only by raising users' awareness of the limitations of metabarcoding data and DNA reference databases will adequate interpretations of these data be achieved.

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crabs—A software program to generate curated reference databases for metabarcoding sequencing data

Cited by 21 publications

References 86 publications

Using surface environmental DNA to assess arthropod biodiversity within a forested ecosystem

Using surface environmental DNA to assess arthropod biodiversity within a forested ecosystem

Assessing the utility of marine filter feeders for environmental DNA (eDNA) biodiversity monitoring

Navigating the seven challenges of taxonomic reference databases in metabarcoding analyses

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