Marine Sediment Sample Pre-processing for Macroinvertebrates Metabarcoding: Mechanical Enrichment and Homogenization

Aylagas, Eva; Mendibil, Iñaki; Borja, Á.; Rodríguez-Ezpeleta, Naiara

doi:10.3389/fmars.2016.00203

Cited by 37 publications

(41 citation statements)

References 29 publications

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“…Sequencing costs are likely to decline in the future and could be further decreased by pooling more samples in each sequencing run and by pre-sorting samples according to biomass (e.g. using sieves Leray & Knowlton 2015;Aylagas et al 2016b;Elbrecht, Peinert & Leese 2017). All laboratory steps from DNA extraction to library preparation currently accumulate to 70€ per sample, leading to a total cost of 180€ per sample in this study, similar to previous estimates (Ji et al 2013;Stein et al 2014).…”

Section: Discussionsupporting

confidence: 75%

Assessing strengths and weaknesses of DNA metabarcoding based macroinvertebrate identification for routine stream monitoring

Elbrecht

Vamos

Meissner

et al. 2017

Preprint

100

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18 19 1) DNA metabarcoding holds great promise for the assessment of macroinvertebrates in stream ecosystems. However, few 20 large-scale studies have compared the performance of DNA metabarcoding with that of routine morphological identification. 212) We performed metabarcoding using four primer sets on macroinvertebrate samples from 18 stream sites across Finland. 22The samples were collected in 2013 and identified based on morphology as part of a Finnish stream monitoring program. 23Specimens were morphologically classified, following standardised protocols, to the lowest taxonomic level for which 24 identification was feasible in the routine national monitoring. 253) DNA metabarcoding identified more than twice the number of taxa than the morphology-based protocol, and also yielded 26 a higher taxonomic resolution. For each sample, we detected more taxa by metabarcoding than by the morphological 27 method, and all four primer sets exhibited comparably good performance. Sequence read abundance and the number of 28 specimens per taxon (a proxy for biomass) were significantly correlated in each sample, although the adjusted R 2 were low. 29With a few exceptions, the ecological status assessment metrics calculated from morphological and DNA metabarcoding 30 datasets were similar. Given the recent reduction in sequencing costs, metabarcoding is currently approximately as 31 expensive as morphology-based identification. 32 4) Using samples obtained in the field, we demonstrated that DNA metabarcoding can achieve comparable assessment 33 results to current protocols relying on morphological identification. Thus, metabarcoding represents a feasible and reliable 34 method to identify macroinvertebrates in stream bioassessment, and offers powerful advantage over morphological 35 identification in providing identification for taxonomic groups that are unfeasible to identify in routine protocols. To unlock 36 the full potential of DNA metabarcoding for ecosystem assessment, however, it will be necessary to address key problems 37 with current laboratory protocols and reference databases. 38 39 40

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Section: Discussionsupporting

confidence: 75%

Assessing strengths and weaknesses of DNA metabarcoding based macroinvertebrate identification for routine stream monitoring

Elbrecht

Vamos

Meissner

et al. 2017

Preprint

100

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“…Sediment samples (3 samples per site) were collected by sieving material collected from a Van Veen grab (0.07–0.1 m 2 ) with a 1‐mm‐mesh size sieve. Retained material was processed following Aylagas, Borja, et al () and Aylagas, Mendibil, et al () based on decantation and homogenization phases, and retrieved benthic macroinvertebrates were stored in 96% ethanol at −20°C. Homogenization was done by blending the decanted organic material in a PHILIPS hr2095 700 W 2‐L glass jar with 96% ethanol until no fragments of animals and other organic materials could be observed.…”

Section: Methodsmentioning

confidence: 99%

Considerations for metabarcoding‐based port biological baseline surveys aimed at marine nonindigenous species monitoring and risk assessments

Rey

Basurko

Rodríguez-Ezpeleta

2020

Ecology and Evolution

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Monitoring introduction and spread of nonindigenous species via maritime transport and performing risk assessments require port biological baseline surveys. Yet, the comprehensiveness of these surveys is often compromised by the large number of habitats present in a port, the seasonal variability, and the time‐consuming morphological approach used for taxonomic identification. Metabarcoding represents a promising alternative for rapid comprehensive port biological baseline surveys, but its application in this context requires further assessments. We applied metabarcoding (based on barcodes of the cytochrome c oxidase subunit I and of the 18S ribosomal RNA gene) to 192 port samples collected (a) from diverse habitats (water column—including environmental DNA and zooplankton, sediment, and fouling structures), (b) at different sites (from inner to outer estuary), and iii) during the four seasons of the year. By comparing the biodiversity metrics derived from each sample group, we show that each sampling method resulted in a distinct community profile and that environmental DNA alone cannot substitute for organismal sampling, and that, although sampling at different seasons and locations resulted in higher observed biodiversity, operational results can be obtained by sampling selected locations and seasons. By assessing the taxonomic composition of the samples, we show that metabarcoding data allowed the detection of previously recorded nonindigenous species as well as to reveal presence of new ones, even if in low abundance. Synthesis and application. Our comprehensive assessment of metabarcoding for port biological baseline surveys sets the basics for cost‐effective, standardized, and comprehensive monitoring of nonindigenous species and for performing risk assessments in ports. This development will contribute to the implementation of the recently entered into force International Convention for the Control and Management of Ships' Ballast Water and Sediments.

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“…Analyses of complex community samples has often been based on extraction from the homogenized tissue present in the sample (Aylagas, Mendibil, Borja, & Rodríguez-Ezpeleta, 2016;Emilson et al, 2017;Kocher et al, 2017). As an alternative approach, Hajibabaei, Spall, Shokralla, and van Konynenburg (2012) and others (e.g.…”

Section: Introductionmentioning

confidence: 99%

Establishing arthropod community composition using metabarcoding: Surprising inconsistencies between soil samples and preservative ethanol and homogenate from Malaise trap catches

Marquina

Esparza‐Salas

Roslin

et al. 2019

Molecular Ecology Resources

117

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DNA metabarcoding allows the analysis of insect communities faster and more efficiently than ever before. However, metabarcoding can be conducted through several approaches, and the consistency of results across methods has rarely been studied. We compare the results obtained by DNA metabarcoding of the same communities using two different markers – COI and 16S – and three different sampling methods: (a) homogenized Malaise trap samples (homogenate), (b) preservative ethanol from the same samples, and (c) soil samples. Our results indicate that COI and 16S offer partly complementary information on Malaise trap samples, with each marker detecting a significant number of species not detected by the other. Different sampling methods offer highly divergent estimates of community composition. The community recovered from preservative ethanol of Malaise trap samples is significantly different from that recovered from homogenate. Small and weakly sclerotized insects tend to be overrepresented in ethanol while strong and large taxa are overrepresented in homogenate. For soil samples, highly degenerate COI primers pick up large amounts of nontarget DNA and only 16S provides adequate analyses of insect diversity. However, even with 16S, very little overlap in molecular operational taxonomic unit (MOTU) content was found between the trap and the soil samples. Our results demonstrate that none of the tested sampling approaches is satisfactory on its own. For instance, DNA extraction from preservative ethanol is not a valid replacement for destructive bulk extraction but a complement. In future metabarcoding studies, both should ideally be used together to achieve comprehensive representation of the target community.

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Marine Sediment Sample Pre-processing for Macroinvertebrates Metabarcoding: Mechanical Enrichment and Homogenization

Cited by 37 publications

References 29 publications

Assessing strengths and weaknesses of DNA metabarcoding based macroinvertebrate identification for routine stream monitoring

Assessing strengths and weaknesses of DNA metabarcoding based macroinvertebrate identification for routine stream monitoring

Considerations for metabarcoding‐based port biological baseline surveys aimed at marine nonindigenous species monitoring and risk assessments

Establishing arthropod community composition using metabarcoding: Surprising inconsistencies between soil samples and preservative ethanol and homogenate from Malaise trap catches

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