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| 2. |
- Ahmed, Mohammed, 1986-, et al.
(författare)
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Metabarcoding of soil nematodes: the importance of taxonomiccoverage and availability of reference sequences in choosingsuitable marker(s)
- 2019
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Ingår i: Metabarcoding and Metagenomics. - : Pensoft Publishers. - 2534-9708. ; 3, s. 77-99
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Tidskriftsartikel (refereegranskat)abstract
- For many organisms, there is agreement on the specific genomic region used for developing barcode markers. With nematodes, however, it has been found that the COI region designated for most animals lacks the taxonomic coverage (ability to amplify a diverse group of taxa) required of a metabarcoding marker. For that reason, studies on metabarcoding of nematodes thus far have utilized primarily regions within the highly conserved 18S ribosomal DNA. Two popular markers within this region are the ones flanked by the primer pairs NF1-18Sr2b and SSUF04-SSUR22. The NF1-18Sr2b primer pair, especially, has been critiqued as not being specific enough for nematodes leading to suggestions for other candidate markers while the SSUF04-SSUR22 region has hardly been tested on soil nematodes. The current study aimed to evaluate these two markers against other alternative ones within the 28S rDNA and the COI region for their suitability for nematode metabarcoding. The results showed that the NF1-18Sr2b marker could offer wide coverage and good resolution for characterizing soil nematodes. Sufficient availability of reference sequences for this region was found to be a significant factor that resulted in this marker outperforming the other markers, particularly the 18S-based SSUFO4-SSUR22 marker. None of the other tested regions compared with this marker in terms of the proportion of the taxa recovered. The COI-based marker had the lowest number of taxa recovered, and this was due to the poor performance of its primers and the insufficient number of reference sequences in public databases. In summary, this study highlights how dependent the success of metabarcoding is on the availability of a good reference sequence collection for the marker of choice as well as its taxonomic coverage.
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| 3. |
- Kahlert, Maria
(författare)
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Metadata standards and practical guidelines for specimen and DNA curation when building barcode reference libraries for aquatic life
- 2021
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Ingår i: Metabarcoding and Metagenomics. - : Pensoft Publishers. - 2534-9708. ; 5, s. 17-33
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Tidskriftsartikel (refereegranskat)abstract
- DNA barcoding and metabarcoding is increasingly used to effectively and precisely assess and monitor biodiversity in aquatic ecosystems. As these methods rely on data availability and quality of barcode reference libraries, it is important to develop and follow best practices to ensure optimal quality and traceability of the metadata associated with the reference barcodes used for identification. Sufficient metadata, as well as vouchers, corresponding to each reference barcode must be available to ensure reliable barcode library curation and, thereby, provide trustworthy baselines for downstream molecular species identification. This document (1) specifies the data and metadata required to ensure the relevance, the accessibility and traceability of DNA barcodes and (2) specifies the recommendations for DNA harvesting and for the storage of both voucher specimens/samples and barcode data.
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| 4. |
- Kahlert, Maria, et al.
(författare)
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Molecular versus morphological data for benthic diatoms biomonitoring in Northern Europe freshwater and consequences for ecological status
- 2019
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Ingår i: Metabarcoding and Metagenomics. - : Pensoft Publishers. - 2534-9708. ; 3, s. 21-35
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Tidskriftsartikel (refereegranskat)abstract
- Diatoms are known to be efficient bioindicators for water quality assessment because of their rapid response to environmental pressures and their omnipresence in water bodies. The identification of benthic diatoms communities in the biofilm, coupled with quality indices such as the Indice de polluosensibilité spécifique (IPS) can be used for biomonitoring purposes in freshwater. However, the morphological identification and counting of diatoms species under the microscope is time-consuming and requires extensive expertise to deal with a constantly evolving taxonomy. In response, a molecular-based and potentially more cost-effective method has been developed, coupling high-throughput sequencing and DNA metabarcoding. The method has already been tested for water quality assessment with diatoms in Central Europe. In this study, we applied both the traditional and molecular methods on 180 biofilms samples from Northern Europe (rivers and lakes of Fennoscandia and Iceland). The DNA metabarcoding data were obtained on two different DNA markers, the 18S-V4 and rbcL barcodes, with the NucleoSpin Soil kit for DNA extraction and sequenced on an Ion Torrent PGM platform. We assessed the ability of the molecular method to produce species inventories, IPS scores and ecological status class comparable to the ones generated by the traditional morphology-based approach. The two methods generated correlated but significantly different IPS scores and ecological status assessment. The observed deviations are explained by presence/absence and abundance discrepancies in the species inventories, mainly due to the incompleteness of the barcodes reference databases, primer bias and strictness of the bioinformatic pipeline. Abundance discrepancies are less common than presence/absence discrepancies but have a greater effect on the ecological assessment. Missing species in the reference databases are mostly acidophilic benthic diatoms species, typical of the low pH waters of Northern Europe. The two different DNA markers also generated significantly different ecological status assessments. The use of the 18S-V4 marker generates more species inventories discrepancies, but achieves an ecological assessment more similar to the traditional morphology-based method. Further development of the metabarcoding method is needed for its use in environmental assessment. For its application in Northern Europe, completion and curation of reference databases are necessary, as well as evaluation of the currently available bioinformatics pipelines. New indices, fitted for environmental biomonitoring, should also be developed directly from molecular data.
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| 5. |
- Kahlert, Maria
(författare)
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Recommendations for the preservation of environmental samples in diatom metabarcoding studies
- 2022
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Ingår i: Metabarcoding and Metagenomics. - : Pensoft Publishers. - 2534-9708. ; 6, s. 349-365
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Tidskriftsartikel (refereegranskat)abstract
- Implementation of DNA metabarcoding for diatoms for environmental monitoring is now moving from a research to an operational phase, requiring rigorous guidelines and standards. In particular, the first steps of the diatom metabarcoding process, which consist of sampling and storage, have been addressed in various ways in scientific and pilot studies and now need to be rationalised. The objective of this study was to compare three currently applied preservation protocols through different storage durations (ranging from one day to one year) for phytobenthos and phytoplankton samples intended for diatom DNA metabarcoding analysis. The experimental design used samples from four freshwater and two marine sites of diverse ecological characteristics. The impact of the sample preservation and storage duration was assessed through diatom metabarcoding endpoints: DNA quality and quantity, diversity and richness, diatom assemblage composition and ecological index values (for freshwater samples). The yield and quality of extracted DNA only decreased for freshwater phytobenthos samples preserved with ethanol. Diatom diversity was not affected and their taxonomic composition predominantly reflected the site origin. Only rare taxa (< 100 reads) differed among preservation methods and storage durations. For biomonitoring purposes, freshwater ecological index values were not affected by the preservation method and storage duration tested (including ethanol preservation), all treatments returning the same ecological status for a site. This study contributes to consolidating diatom metabarcoding. Thus, accompanied by operational standards, the method will be ready to be confidently deployed and prescribed in future regulatory monitoring.
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| 6. |
- Kahlert, Maria
(författare)
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A strategy for successful integration of DNA-based methods in aquatic monitoring
- 2022
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Ingår i: Metabarcoding and Metagenomics. - : Pensoft Publishers. - 2534-9708. ; 6, s. 215-226
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Tidskriftsartikel (refereegranskat)abstract
- Recent advances in molecular biomonitoring open new horizons for aquatic ecosystem assessment. Rapid and cost-effective methods based on organismal DNA or environmental DNA (eDNA) now offer the opportunity to produce inventories of indicator taxa that can subsequently be used to assess biodiversity and ecological quality. However, the integration of these new DNA-based methods into current monitoring practices is not straightforward, and will require coordinated actions in the coming years at national and international levels. To plan and stimulate such an integration, the European network DNAqua-Net (COST Action CA15219) brought together international experts from academia, as well as key environmental biomonitoring stakeholders from different European countries. Together, this transdisciplinary consortium developed a roadmap for implementing DNA-based methods with a focus on inland waters assessed by the EU Water Framework Directive (2000/60/EC). This was done through a series of online workshops held in April 2020, which included fifty participants, followed by extensive synthesis work. The roadmap is organised around six objectives: 1) to highlight the effectiveness and benefits of DNA-based methods, 2) develop an adaptive approach for the implementation of new methods, 3) provide guidelines and standards for best practice, 4) engage stakeholders and ensure effective knowledge transfer, 5) support the environmental biomonitoring sector to achieve the required changes, 6) steer the process and harmonise efforts at the European level. This paper provides an overview of the forum discussions and the common European views that have emerged from them, while reflecting the diversity of situations in different countries. It highlights important actions required for a successful implementation of DNA-based biomonitoring of aquatic ecosystems by 2030.
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| 7. |
- Magesh, Shruthi, et al.
(författare)
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Mumame: A software tool for quantifying gene-specific point-mutations in shotgun metagenomic data
- 2019
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Ingår i: Metabarcoding and Metagenomics. - : Pensoft Publishers. - 2534-9708. ; 3
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Tidskriftsartikel (refereegranskat)abstract
- Metagenomics has emerged as a central technique for studying the structure and function of microbial communities. Often the functional analysis is restricted to classification into broad functional categories. However, important phenotypic differences, such as resistance to antibiotics, are often the result of just one or a few point mutations in otherwise identical sequences. Bioinformatic methods for metagenomic analysis have generally been poor at accounting for this fact, resulting in a somewhat limited picture of important aspects of microbial communities. Here, we address this problem by providing a software tool called Mumame, which can distinguish between wildtype and mutated sequences in shotgun metagenomic data and quantify their relative abundances. We demonstrate the utility of the tool by quantifying antibiotic resistance mutations in several publicly available metagenomic data sets. We also identified that sequencing depth is a key factor to detect rare mutations. Therefore, much larger numbers of sequences may be required for reliable detection of mutations than for most other applications of shotgun metagenomics. Mumame is freely available online (http://microbiology.se/software/mumame).
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| 8. |
- Marquina, Daniel, 1989-, et al.
(författare)
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Evaluation of non-destructive DNA extraction protocols for insect metabarcoding : gentler and shorter is better
- 2022
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Ingår i: Metabarcoding and Metagenomics. - : Pensoft Publishers. - 2534-9708. ; 6, s. 187-201
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Tidskriftsartikel (refereegranskat)abstract
- DNA metabarcoding can accelerate research on insect diversity, as it is cheap and fast compared to manual sorting and identification. Most metabarcoding protocols require homogenisation of the sample, preventing further work on the specimens. Mild digestion of the tissue by incubation in a lysis buffer has been proposed as an alternative, and, although some mild lysis protocols have already been presented, they have so far not been evaluated against each other. Here, we analyse how two mild lysis buffers (one more aggressive, one gentler in terms of tissue degradation), two different incubation times, and two DNA purification methods (a manual precipitation and an automated protocol) affect the accuracy of retrieving the true composition of mock communities using two mitochondrial markers (COI and 16S). We found that protocol-specific variation in concentration and purity of the DNA extracts produced had little effect on the recovery of species. However, the two lysis treatments differed in quantification of species abundances. Digestion in the gentler buffer and for a shorter time yielded better representation of original sample composition. Digestion in a more aggressive buffer or longer incubation time yielded lower alpha diversity values and increased differences between metabarcoding results and the true species-abundance distribution. We conclude that the details of non-destructive protocols can have a significant effect on metabarcoding performance. A short and mild lysis treatment appears the best choice for recovering the true composition of the sample. This not only improves accuracy, but also comes with a faster processing time than the other treatments.
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| 9. |
- Nilsson, R. Henrik, 1976, et al.
(författare)
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Introducing guidelines for publishing DNA-derived occurrence data through biodiversity data platforms
- 2022
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Ingår i: Metabarcoding and Metagenomics. - : Pensoft Publishers. - 2534-9708. ; 6, s. 239-244
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Tidskriftsartikel (refereegranskat)abstract
- DNA sequencing efforts of environmental and other biological samples disclose unprecedented and largely untapped opportunities for advances in the taxonomy, ecology, and geographical distributions of our living world. To realise this potential, DNA-derived occurrence data (notably sequences with dates and coordinates) – much like traditional specimens and observations – need to be discoverable and interpretable through biodiversity data platforms. The Global Biodiversity Information Facility (GBIF) recently headed a community effort to assemble a set of guidelines for publishing DNA-derived data. These guidelines target the principles and approaches of exposing DNA-derived occurrence data in the context of broader biodiversity data. They cover a choice of terms using a controlled vocabulary, common pitfalls, and good practices, without going into platform-specific details. Our hope is that they will benefit anyone interested in better exposure of DNA-derived occurrence data through general biodiversity data platforms, including national biodiversity portals. This paper provides a brief rationale and an overview of the guidelines, an up-to-date version of which is maintained at https://doi.org/10.35035/doc-vf1a-nr22. User feedback and interaction are encouraged as new techniques and best practices emerge.
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| 10. |
- Roslin, Tomas
(författare)
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Ecological signals of arctic plant-microbe associations are consistent across eDNA and vegetation surveys
- 2023
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Ingår i: Metabarcoding and Metagenomics. - 2534-9708. ; 7, s. 155-193
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Tidskriftsartikel (refereegranskat)abstract
- Understanding how different taxa respond to abiotic characteristics of the environment is of key interest for understanding the assembly of communities. Yet, whether eDNA data will suffice to accurately capture environmental imprints has been the topic of some debate. In this study, we characterised patterns of species occurrences and co-occurrences in Zackenberg in northeast Greenland using environmental DNA. To explore the potential for extracting ecological signals from eDNA data alone, we compared two approaches (visual vegetation surveys and soil eDNA metabarcoding) to describing plant communities and their responses to abiotic conditions. We then examined plant associations with microbes using a joint species distribution model. We found that most (68%) of plant genera were detectable by both vegetation surveys and eDNA signatures. Species-specific occurrence data revealed how plants, bacteria and fungi responded to their abiotic environment - with plants, bacteria and fungi all responding similarly to soil moisture. Nonetheless, a large proportion of fungi decreased in occurrences with increasing soil temperature. Regarding biotic associations, the nature and proportion of the plant-microbe associations detected were consistent between plant data identified via vegetation surveys and eDNA. Of pairs of plants and microbe genera showing statistically supported associations (while accounting for joint responses to the environment), plants and bacteria mainly showed negative associations, whereas plants and fungi mainly showed positive associations. Ample ecological signals detected by both vegetation surveys and by eDNA-based methods and a general correspondence in biotic associations inferred by both methods, suggested that purely eDNA-based approaches constitute a promising and easily applicable tool for studying plant-soil microbial associations in the Arctic and elsewhere.
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