Bioinformatic pipelines combining denoising and clustering tools allow for more comprehensive prokaryotic and eukaryotic metabarcoding

Brandt, Miriam I.; Trouche, Blandine; Quintric, Laure; Günther, Babett; Wincker, Patrick; Poulain, Julie; Arnaud‐Haond, Sophie

doi:10.1111/1755-0998.13398

Cited by 62 publications

(48 citation statements)

References 108 publications

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“…All primers were synthesized with Illumina adapters and later combined with Illumina barcodes to allow multiplexing. Compared to these original primer sequences, Inosine (I) were changed with ''wobbles'' (N) to create degenerate primers compatible with the Brandt et al (2021) were as follows: 98 • C for 30 s, followed by a specific number of cycles of 98 • C for 10 s, annealing for 45 s, and 72 • C for 60 s, with a final elongation at 72 • C for 10 min. For 18S specifically, an annealing temperature of 50 • C and 40 cycles were applied, and the corresponding conditions for COI were 48 • C and 40 cycles.…”

Section: Dna Extraction and Sequencingmentioning

confidence: 99%

Metabarcoding confirms the opportunistic foraging behaviour of Atlantic bluefin tuna and reveals the importance of gelatinous prey

Günther

Fromentin

Métral

et al. 2021

PeerJ

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Studies of the diet, feeding habits and trophic activity of top marine predators are essential for understanding their trophodynamics. The main direct method used for such studies thus far has been morphological inventories of stomach contents. This approach presents limitations such as missing gelatinous prey, which are usually digested too quickly to be detectable. Here, we analysed the stomachs of 48 Atlantic bluefin tuna (Thunnus thynnus, approximately 15 to 60 kg, including juveniles and adult fishes) collected from the Mediterranean Sea through the metabarcoding of two gene regions (cytochrome c oxidase subunit I (COI) and the ribosomal 18S-V1V2 region). The identified prey taxa and their relative read abundances (RRAs) estimated using COI results were in line with the findings of morphologically based inventories simultaneously performed on the same set of tuna samples. In both cases (and with the same rankings), the prey taxa included anchovy (Engraulis encrasicolus, here detected in more than 80% of samples, RRA = 43%), sardine (Sardina pilchardus, also approximately 80%, RRA = 30%), sprat (Sprattus sprattus, approximately 66%, RRA = 8%), mackerel (Scomber colias, approximately 44%, RRA = 7%) and cephalopods (approximately 15%, RRA = 1.4%). Another striking result was the detection, based on 18S (with which vertebrates were detected as the most abundant group, RRA = 61.6%), of a high prevalence and diversity of gelatinous organisms (RRA = 27.1%), including cnidarians (6.7%), salps (11.7%), and ctenophores (8.7%), the latter increasing with the size of the predator. These results thus support the hypothesis of the role of gelatinous prey in the diet of Atlantic bluefin tuna, suggesting that this species is even more generalist and opportunistic than previously thought. This study further confirms that DNA metabarcoding can be a powerful tool for assessing the diet and trophodynamics of top marine predators.

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Section: Dna Extraction and Sequencingmentioning

confidence: 99%

Metabarcoding confirms the opportunistic foraging behaviour of Atlantic bluefin tuna and reveals the importance of gelatinous prey

Günther

Fromentin

Métral

et al. 2021

PeerJ

Self Cite

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“…Using a multi-species mock sample with known haplotypes based on Sanger sequencing of the highly variable mtCOI gene region, we presented the feasibility and limitations of using DNA metabarcoding data to extract intraspecific genetic diversity information by denoising the sequences into ZOTUs. Most recent studies inferred intraspecific diversity from macroinvertebrate DNA metabarcoding data were also based on denoising algorithms (e.g., Elbrecht et al, 2018;Turon et al, 2019;Brandt et al, 2021).…”

Section: Discussionmentioning

confidence: 99%

Haplotype-level metabarcoding of freshwater macroinvertebrate species: a prospective tool for population genetic analysis

Serrana

Watanabe

2021

Preprint

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The development and evaluation of DNA metabarcoding protocols for haplotype-level resolution require attention, specifically for population genetic analysis, i.e., parallel estimation of genetic diversity and dispersal patterns among multiple species present in a bulk sample. Further exploration and assessment of the laboratory and bioinformatics strategies are warranted to unlock the potential of metabarcoding-inferred population genetic analysis. Here, we assessed the inference of freshwater macroinvertebrate haplotypes from DNA metabarcoding data using mock samples with known Sanger-sequenced haplotypes. We also examined the influence of different DNA template concentrations and PCR cycles on detecting true haplotypes and the reduction of spurious haplotypes obtained from DNA metabarcoding. We tested our haplotyping strategy on a mock sample containing 20 specimens from four species with known haplotypes based on the 658-bp Folmer region of the mitochondrial cytochrome c oxidase gene. The read processing and denoising step resulted in 14 zero-radius operational taxonomic units (ZOTUs) of 421-bp length, with 12 ZOTUs having 100% match with 12 of the Sanger haplotype sequences. Quality passing reads relatively increased with increasing PCR cycles, and the relative abundance of each ZOTUs was consistent for each cycle number. This suggests that increasing the cycle number from 24 to 64 did not affect the relative abundance of quality passing filter reads of each ZOTUs. Our study demonstrated the ability of DNA metabarcoding to infer intraspecific variability while highlighting the challenges that need to be addressed before its possible applications to population genetic studies.

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“…Correction algorithms are increasingly popular as they effectively remove sequencing errors and increase reproducibility, conceptual simplicity, and streamlined processing (Callahan et al 2017). However, recent literature suggests that both molecular entities are complementary rather than mutually exclusive, ASVs allowing describing diversity at the intraspecies level, OTUs at the interspecies level (Turon et al 2020;Brandt et al 2021b). Spurious molecular units are a serious issue in metabarcoding, and even after stringent quality-filtering steps (incl.…”

Section: Genomic Fingerprinting By Bulk or Environmental Dna Metabarcodingmentioning

confidence: 99%

“…The limitations in taxonomic assignment quality are therefore mostly due to the limited amount of data available rather than algorithms, and this is especially true for understudied ecosystems such as the deep-sea. Arbitrarily large and uneven databases containing sequences that have not been truncated to the target sequence length, can decrease the number of accurate taxonomic assignments (Werner et al 2012;Ritari et al 2015;Edgar 2018;Macheriotou et al 2018), and recent work shows that taxonomic assignments based on public databases remain poor for deep-sea metabarcoding data generated with "universal" primers (Brandt et al 2021b). For this reason, the development of smaller, taxon-specific and ecosystem-specific reference databases will be necessary to obtain reliable taxonomic information from metabarcoding studies performed in poorly characterized areas such as the CCFZ.…”

Section: Genomic Fingerprinting By Bulk or Environmental Dna Metabarcodingmentioning

confidence: 99%

Toward a reliable assessment of potential ecological impacts of deep‐sea polymetallic nodule mining on abyssal infauna

Lins

Zeppilli

Menot

et al. 2021

Limnology & Ocean Methods

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The increasing demand for metals is pushing forward the progress of deep-sea mining industry. The abyss between the Clarion and Clipperton Fracture Zones (CCFZ), a region holding a higher concentration of minerals than land deposits, is the most targeted area for the exploration of polymetallic nodules worldwide, which may likely disturb the seafloor across large areas and over many years. Effects from nodule extraction cause acute biodiversity loss of organisms inhabiting sediments and polymetallic nodules. Attention to deep-sea ecosystems and their services has to be considered before mining starts but the lack of basic scientific knowledge on the methodologies for the ecological surveys of fauna in the context of deep-sea mining impacts is still scarce. We review the methodology to sample, process and investigate metazoan infauna both inhabiting sediments and nodules dwelling on these polymetallic-nodule areas. We suggest effective procedures for sampling designs, devices and methods involving gear types, sediment processing, morphological and genetic identification including metabarcoding and proteomic fingerprinting, the assessment of biomass, functional traits, fatty acids, and stable isotope studies within the CCFZ based on both first-hand experiences and literature. We recommend multi-and boxcorers for the quantitative assessments of meio-and macrofauna, respectively. The assessment of biodiversity at species level should be focused and/or the combination of morphological with metabarcoding or proteomic fingerprinting techniques. We highlight that biomass, functional traits, and trophic markers may provide critical insights for biodiversity assessments and how statistical modeling facilitates predicting patterns spatially across point-source data and is essential for conservation management.

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Bioinformatic pipelines combining denoising and clustering tools allow for more comprehensive prokaryotic and eukaryotic metabarcoding

Abstract: S. A flexible pipeline combining clustering and correction tools for prokaryotic and eukaryotic metabarcoding. bioRxiv 717355, ver. 3 peer-reviewed and recommended by PCI Ecology (2020).

Cited by 62 publications

References 108 publications

Metabarcoding confirms the opportunistic foraging behaviour of Atlantic bluefin tuna and reveals the importance of gelatinous prey

Metabarcoding confirms the opportunistic foraging behaviour of Atlantic bluefin tuna and reveals the importance of gelatinous prey

Haplotype-level metabarcoding of freshwater macroinvertebrate species: a prospective tool for population genetic analysis

Toward a reliable assessment of potential ecological impacts of deep‐sea polymetallic nodule mining on abyssal infauna

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