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1,076 results for “Metabarcoding”
Data from: The effect of DNA degradation bias in passive sampling devices on metabarcoding studies of arthropod communities and their associated microbiota
PCR amplification bias is a well-known problem in metagenomic analysis of arthropod communities. In contrast, variation of DNA degradation rates is a largely neglected source of bias. Differential degradation of DNA molecules could cause underrepresentation of taxa in a community sequencing sample. Arthropods are often collected by passive sampling devices, like malaise traps. Specimens in such a trap are exposed to varying periods of suboptimal storage and possibly different rates of DNA degradation. Degradation bias could thus be a significant issue, skewing diversity estimates. Here, we estimate the effect of differential DNA degradation on the recovery of community diversity of Hawaiian arthropods and their associated microbiota. We use a simple DNA size selection protocol to test for degradation bias in mock communities, as well as passively collected samples from actual Malaise traps. We compare the effect of DNA degradation to that of varying PCR conditions, including primer choice, annealing temperature and cycle number. Our results show that DNA degradation does indeed bias community analyses. However, the effect of this bias is of minor importance compared to that induced by changes in PCR conditions. Analyses of the macro and microbiome from passively collected arthropod samples are thus well worth pursuing.
Data from: DNA metabarcoding of Amazonian ichthyoplankton swarms
Tropical rainforests harbor extraordinary biodiversity. The Amazon basin is thought to hold 30% of all river fish species in the world. Information about the ecology, reproduction, and recruitment of most species is still lacking, thus hampering fisheries management and successful conservation strategies. One of the key understudied issues in the study of population dynamics is recruitment. Fish larval ecology in tropical biomes is still in its infancy owing to identification difficulties. Molecular techniques are very promising tools for the identification of larvae at the species level. However, one of their limits is obtaining individual sequences with large samples of larvae. To facilitate this task, we developed a new method based on the massive parallel sequencing capability of next generation sequencing (NGS) coupled with hybridization capture. We focused on the mitochondrial marker cytochrome oxidase I (COI). The results obtained using the new method were compared with individual larval sequencing. We validated the ability of the method to identify Amazonian catfish larvae at the species level and to estimate the relative abundance of species in batches of larvae. Finally, we applied the method and provided evidence for strong temporal variation in reproductive activity of catfish species in the Ucayalí River in the Peruvian Amazon. This new time and cost effective method enables the acquisition of large datasets, paving the way for a finer understanding of reproductive dynamics and recruitment patterns of tropical fish species, with major implications for fisheries management and conservation.
Data from: Evaluation of plant contamination in metabarcoding diet analysis of a herbivore
Fecal DNA metabarcoding is currently used in various fields of ecology to determine animal diets. Contamination of non-food DNA from complex field environments is a considerable challenge to the reliability of this method but has rarely been quantified. We evaluated plant DNA contamination by sequencing the chloroplast trnL P6 loop region from food-controlled geese feces. The average percentage of contaminant sequences per sample was 1.86%. According to the results of generalized linear models, the probability of contamination was highest in samples placed in wet soil. The proportion of contaminant sequences was lowest at the earliest sampling point and was slightly higher in samples placed in open conditions. Exclusion of rare OTUs (operational taxonomic units) was effective for obtaining reliable dietary data from the obtained sequences, and a 1% cutoff reduced the percentage of contaminated samples to less than 30%. However, appropriate interpretation of the barcoding results considering inevitable contamination is an important issue to address. We suggest the following procedures for fecal sampling and sequence data treatment to increase the reliability of DNA metabarcoding diet analyses: (i) Collect samples as soon as possible after deposition, (ii) avoid samples from deposits on wet soil, and (iii) exclude rare OTUs from diet composition estimations.
Data from: How quantitative is metabarcoding: a meta-analytical approach
Metabarcoding has been used in a range of ecological applications such as taxonomic assignment, dietary analysis, and the analysis of environmental DNA. However, after a decade of use in these applications there is little consensus on the extent to which proportions of reads generated corresponds to the original proportions of species in a community. To quantify our current understanding we conducted a structured review and meta-analysis. The analysis suggests that a weak quantitative relationship may exist between the biomass and sequences produced (slope =0.52 ±0.34, p<0.01)), albeit it with a large degree of uncertainty. None of the tested moderators: sequencing platform type, the number of species used in a trial, or the source of DNA were able to explain the variance. Our current understanding of the factors affecting the quantitative performance of metabarcoding is still limited: additional research is required before metabarcoding can be confidently utilised for quantitative applications. Until then, we advocate the inclusion of mock communities when metabarcoding as this facilitates direct assessment of the quantitative ability of any given study.
Data from: 18S rRNA V9 metabarcoding for diet characterization: a critical evaluation with two sympatric zooplanktivorous fish species
The potential of the 18S rRNA V9 metabarcoding approach for diet assessment was explored using MiSeq paired-end (PE; 2 × 150 bp) technology. To critically evaluate the method′s performance with degraded/digested DNA, the diets of two zooplanktivorous fish species from the Bay of Biscay, European sardine (Sardina pilchardus) and European sprat (Sprattus sprattus), were analysed. The taxonomic resolution and quantitative potential of the 18S V9 metabarcoding was first assessed both in silico and with mock and field plankton samples. Our method was capable of discriminating species within the reference database in a reliable way providing there was at least one variable position in the 18S V9 region. Furthermore, it successfully discriminated diet between both fish species, including habitat and diel differences among sardines, overcoming some of the limitations of traditional visual-based diet analysis methods. The high sensitivity and semi-quantitative nature of the 18S V9 metabarcoding approach was supported by both visual microscopy and qPCR-based results. This molecular approach provides an alternative cost and time effective tool for food-web analysis.
Metabarcoding reveals seasonal and spatial patterns of arthropod community assemblages in two contrasting habitats
<p>Illumina reads, representing partial COI barcode. </p> <p>Quality filtered FLS and SLS reads:<br> Samples from oasis<br> FLS reads <br> 150615_I270_FCC7K1NACXX_L4_RSZAKPI005096-37_1.fq.gz<br> 150615_I270_FCC7K1NACXX_L4_RSZAKPI005096-37_2.fq.gz<br> SLS reads<br> 150615_I270_FCC7K1NACXX_L4_RSZAXPI005095-40_1.fq.gz<br> 150615_I270_FCC7K1NACXX_L4_RSZAXPI005095-40_2.fq.gz</p> <p>Samples from desert:<br> FLS reads<br> 150615_I270_FCC7K1NACXX_L4_RSZAKPI005098-39_1.fq.gz<br> 150615_I270_FCC7K1NACXX_L4_RSZAKPI005098-39_2.fq.gz<br> SLS reads<br> 150615_I270_FCC7K1NACXX_L4_RSZAXPI005097-41_1.fq.gz<br> 150615_I270_FCC7K1NACXX_L4_RSZAXPI005097-41_2.fq.gz</p>
Figure 1 from: Anslan S, Nilsson RH, Wurzbacher C, Baldrian P, Tedersoo L, Bahram M (2018) Great differences in performance and outcome of high-throughput sequencing data analysis platforms for fungal metabarcoding. MycoKeys 39: 29-40. https://doi.org/10.3897/mycokeys.39.28109
Figure 1 - Outline of workflow in different analysis pipelines.
Figure 1 from: Bowser ML, Burr SJ, Davis I, Dubois GD, Graham EE, Moan JE, Swenson SW (2019) A test of metabarcoding for Early Detection and Rapid Response monitoring for non-native forest pest beetles (Coleoptera). Research Ideas and Outcomes 5: e48536. https://doi.org/10.3897/rio.5.e48536
Figure 1 Map of sampling locations generated using SimpleMappr (Shorthouse 2010).
Data from: Veronica officinalis product authentication using DNA metabarcoding and HPLC-MS reveals widespread adulteration with Veronica chamaedrys
Studying herbal products derived from local and traditional knowledge and their value chains is one of the main challenges in ethnopharmacology. The majority of these products have a long history of use, but non-harmonized trade and differences in regulatory policies between countries impact their value chains and lead to concerns over product efficacy, safety and quality. Veronica officinalis L. (common speedwell), a member of Plantaginaceae family, has a long history of use in European traditional medicine, mainly in central eastern Europe and the Balkans. However, no specified control tests are available either to establish the quality of derived herbal products or for the discrimination of its most common substitute, V. chamaedrys L. (germander speedwell). In this study, we use DNA metabarcoding and high performance liquid chromatography coupled with mass spectrometry (HPLC-MS) to authenticate sixteen V. officinalis herbal products and compare the potential of the two approaches to detect substitution, adulteration and the use of unreported constituents. HPLC-MS showed high resolution in detecting phytochemical target compounds, but did not enable detection of specific plant species in the products. DNA metabarcoding detected V. officinalis in only 15% of the products, whereas it detected V. chamaedrys in 62% of the products. The results confirm that DNA metabarcoding can be used to test for the presence of Veronica species, and detect substitution and/or admixture of other Veronica species, as well as simultaneously detect all other species present. Our results confirm that none of the herbal products contained exactly the species listed on the label, and all included substitutes, contaminants or fillers. This study highlights the need for authentication of raw herbals along the value chain of these products. An integrative methodology can assess both the quality of herbal products in terms of target compound concentrations and species composition, as well as admixture and substitution with other chemical compounds and plants.
Data from: Environmental DNA metabarcoding of wild flowers reveals diverse communities of terrestrial arthropods
Terrestrial arthropods comprise the most species rich communities on Earth, and grassland flowers provide resources for hundreds of thousands of species. Diverse grassland ecosystems worldwide are threatened by various types of environmental change, which has led to decline in arthropod diversity, while monitoring grassland arthropod diversity is time consuming and strictly dependent on declining taxonomic expertise. Environmental DNA (eDNA) metabarcoding of complex samples has demonstrated that information on species compositions can be efficiently and non-invasively obtained. Here, we test the potential of wild flowers as a novel source of arthropod eDNA. We performed eDNA metabarcoding of flowers from several different plant species using two sets of generic primers, targeting the mitochondrial genes 16S rRNA and COI. Our results show that terrestrial arthropod species leave traces of DNA on the flowers that they interact with. We obtained eDNA from at least 135 arthropod species in 67 families and 14 orders, together representing diverse ecological groups including pollinators, parasitoids, gall inducers, predators and phytophagous species. Arthropod communities clustered together according to plant species. Our data also indicate that this experiment was not exhaustive, and that an even higher arthropod richness could be obtained using this eDNA approach. Overall, our results demonstrate that it is possible to obtain information on diverse communities of insects and other terrestrial arthropods from eDNA metabarcoding of wild flowers. This novel source of eDNA represents a vast potential for addressing fundamental research questions in ecology, obtaining data on cryptic and unknown species of plant-associated arthropods, as well as applied research on pest management or conservation of endangered species such as wild pollinators.
Figure 3 from: Riit T, Tedersoo L, Drenkhan R, Runno-Paurson E, Kokko H, Anslan S (2016) Oomycete-specific ITS primers for identification and metabarcoding. MycoKeys 14: 17-30. https://doi.org/10.3897/mycokeys.14.9244
Figure 3 - Fraction of oomycete reads in individual soil samples.
Data from: Direct PCR meets high-throughput sequencing - metabarcoding of chironomid communities without DNA extraction
<p>Abstract</p> <p>Metabarcoding is a valuable tool for investigating insect community compositions. However, high-throughput applications, such as for biomonitoring, require cost-effective and user-friendly procedures. To investigate if the time-consuming and labour-intensive DNA isolation step can be omitted in metabarcoding, we studied the difference in detection rates and individual read abundance using standard DNA isolation versus direct PCR protocols. Metabarcoding with and without DNA isolation was performed on artificially created communities with known composition as well as on natural communities both of the dipteran family Chironomidae to compare detection rates, individual read abundances and presence-absence community composition. The data sets include read abundances of all artificial and natural community samples. Compositions of the samples per data set are described in the respective README files. ASVs/OTUs and their respective DNA sequences are given. R Scripts for bioinformatic processing (dada2 for ASVs, JAMP for OTUs) are provided.</p> <p>Methods</p> <p>Chironomidae were retrieved from artificial ponds of the Eußerthal Ecosystem Research Station (EERES) near Landau, Germany, in 2019 and 2020. Adult specimens were collected from passive emergence traps. Chironomid samples were stored in 70% ethanol and later dried at 60°C. Samples were then finely ground using a bead mill. PCR-grade water was added to each tissue sample and thoroughly vortexed. The tissue-water mixes were frozen at -20°C until further analysis. Artificial communities were created by pipetting tissue-water mixes of individual chironomids. Natural communities from eight ponds and five consecutive sampling dates were selected to assess the applicability of the dPCR approach compared to standard metabarcoding protocols on natural chironomid communities. Four of the artificial ponds were treated with the mosquito control agent <em>Bacillus thuringiensis israelensis</em> (Bti). Tissue-water mixes of artificial and natural communities were both directly applied to PCR and used for DNA isolation. Illumina sequencing was performed and raw data were bioinformatically prepared. For more details see "Direct PCR meets high-throughput sequencing - metabarcoding of chironomid communities without DNA extraction" (Röder & Schwenk 2023). Raw sequences are available through GenBank SRA archive (BioProject accession number PRJNA989176). </p>
Evaluation of Next-Generation Sequencing-based Metabarcoding Versus Culturing for Microbiological Assessment in Infectious Keratitis
ClinicalTrials.gov study NCT05183906. IPD Sharing: NO. Countries: 1. Publications: 0.
Data from: How quantitative is metabarcoding: a meta-analytical approach
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Data from: DNA metabarcoding of Amazonian ichthyoplankton swarms
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Data from: Environmental DNA metabarcoding of wild flowers reveals diverse communities of terrestrial arthropods
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Data from: 18S rRNA V9 metabarcoding for diet characterization: a critical evaluation with two sympatric zooplanktivorous fish species
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Data from: DNA metabarcoding reveals diet overlap between the endangered Walia ibex and domestic goats - implications for conservation
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Data from: The effect of DNA degradation bias in passive sampling devices on metabarcoding studies of arthropod communities and their associated microbiota
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Data from: A broadly applicable COI primer pair and an efficient single‐tube amplicon library preparation protocol for metabarcoding
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