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Environmental DNA, or eDNA, methodologies can enable the rapid detection of a target species without either visual or physical confirmation of the species presence. Over the last decade, targeted quantitative PCR (qPCR) assays have become an increasingly useful method employed by government and non-government agencies alike for purposes such as protecting and preserving ecosystems from invasive species, or for the conservation of endangered species. As the application of eDNA to answer ecological questions pushes the limits of qPCR-based detection, there is a pressing need to standardise the way qPCR results are reported and interpreted, as well as the way qPCR assays are evaluated for use outside of the remit of the original study.Natural England is one such government agency who have begun to use eDNA methodologies more widely to answer ecological questions. However, while some qPCR assays available for detecting the presence or absence of species such as the great crested newt (Triturus cristatus) have been specified, validated and quality assured to a high degree (Biggs et al. 2014), existing qPCR assays for other species are generally less well developed and validated. Additionally, for some species there are multiple qPCR assays available, with each being developed and validated to different stages. As such, Natural England identified a need to understand how the data derived from eDNA should be interpreted dependent on the level of qPCR assay development, and ultimately the confidence they can have in the accuracy of resulting data, including the associated risk of false positives or false negatives.NatureMetrics has developed a prototype web-based tool and protocol (European Technical Readiness Level 5) which would enable end users such as Natural England to inform their interpretation of qPCR results. The prototype is currently in the beta testing stage and is expected to be available in the coming months. The web-based tool will simplify qPCR assay evaluation, enabling end users to select the most appropriate qPCR assay for their needs, as well as standardise the reporting and interpretation of their qPCR results by generating a report at both the sample and site level from the inputted qPCR data.

The overall goal of the EnvMetaGen project No 668981 is to expand the research and innovation potential of InBIO – Research network in Biodiversity and Evolutionary Biology, through the creation of an ERA Chair in Environmental Metagenomics. This field was selected as the focus of the ERA Chair, because Environmental DNA (eDNA) analysis is increasingly being used for biodiversity assessment, diet analysis, detection of rare or invasive species, population genetics, and ecosystem functional analysis. In this context, the work plan of EnvMetaGen includes one work package (WP) dedicated to the Deployment of an eDNA Lab (WP4), which involves the training of InBIO researchers and technicians for implementing best practice protocols for the analysis of eDNA (Task 4.2). This report provides an overview of best practices for analysing eDNA samples, focusing on vertebrate faeces, water samples and bulks of invertebrates. It covers all steps from DNA extraction to amplification and sequencing in high throughput platforms, while addressing the challenges identified in each step. Moreover, it describes protocols already optimised and currently under development at InBIO, with particular reference to the detection of single species, diet assessments and biodiversity assessments. These three different outputs are fully aligned with the three key application areas that have been targeted in EnvMetaGen for a wider application of environmental metagenomics, and on which the development of strategic triple helix initiatives will focus on. These will therefore contribute to mainstream this technology as a new cost-effective solution to tackle current challenges faced by business partners and other stakeholders, while fostering InBIO-Industry-Government relationships (WP5 - Strengthening the triple helix). The protocols described herein were developed in close connection to, and build on accomplishments of, the other WPs in the project, including: i) the recruitment of the ERA Chair team (WP2), ii) secondments and researcher exchanges through collaborations with international networks (WP3), iii) the enhancement of the computational infrastructure at InBIO (WP4), and iv) participation in and organization of workshops and conferences (WP6). Future directions in eDNA analyses at InBIO, and other potential laboratory pipelines for future optimisation are identified. Together, Deliverables D4.2-D4.5 (this document; Egeter et al. 2018; Ferreira et al. 2018; Galhardo et al. 2018) form a detailed account of the successful deployment of a fully functional eDNA lab under the EnvMetaGen project, and provide a valuable resource for eDNA practitioners in all spheres of the triple-helix model.

The overall goal of the EnvMetaGen project No 668981 is to expand the research and innovation potential of InBIO – Research network in Biodiversity and Evolutionary Biology - through the creation of an ERA Chair in Environmental Metagenomics. This field was selected as the focus of the ERA Chair, because Environmental DNA (eDNA) analysis is increasingly being used for biodiversity assessment, diet analysis, detection of rare or invasive species, population genetics and ecosystem functional analysis. In this context, the work plan of EnvMetaGen includes one work package dedicated to the Deployment of an eDNA Lab (WP4), which involves the training of InBIO researchers and technicians for implementing best practice protocols for the analysis of eDNA (Task 4.2). These protocols are essential to the development of research projects in association with business partners and other stakeholders in key application areas identified in the project, and thus to the strengthening of InBIO triple-helix initiatives (InBIO-Industry-Government; WP5). This report (Deliverable D4.5) builds upon previous ones (Deliverables D4.2-D4.4, respectively Ferreira et al. (2018), Egeter et al. (2018), Paupério et al. (2018)) and provides an overview of the processing protocols for DNA sequence data generated by next-gen platforms within EnvMetaGen-affiliated projects. Deliverables D4.2-D4.5 form a detailed account of the successful deployment of a fully functional eDNA lab under the EnvMetaGen project and provide a valuable resource for eDNA practitioners in all spheres of the triple-helix model. This development was made possible through the recruitment of the ERA Chair team (WP2), secondments and Junior Researcher exchanges through the collaboration with international networks (WP3), an enhancement of computational infrastructure at InBIO (WP4) and participation of team members in workshops and conferences (WP6).

The overall goal of ERA Chair/EnvMetaGen project No 668981 is to expand the research and innovation potential of InBIO – Research network in Biodiversity and Evolutionary Biology, through the creation of an ERA Chair in Environmental Metagenomics. This field was selected as the focus of the ERA Chair, because Environmental DNA (eDNA) analysis is increasingly being used for biodiversity assessment, diet analysis, detection of rare or invasive species, population genetics and ecosystem functional analysis. In this context, the work plan of EnvMetaGen includes one work package dedicated to the Deployment of an eDNA Lab (WP4), which involves the training of InBIO researchers and technicians for implementing best practice protocols for the analysis of eDNA (Task 4.2). These protocols are essential for key application areas and to the development of research projects in association with business partners and other stakeholders, and thus to the strengthening of InBIO triple-helix initiatives (InBIO-Industry-Government; WP5). This report provides an overview of the current state of the art for collecting and preserving eDNA samples, with particular focus on vertebrate faecal samples, water samples and bulk invertebrate samples, which have been selected as key targets for the development of triple helix strategic initiatives (Task 5.3). The protocols already optimized and currently under development for the collection and preservation of eDNA samples are reported herein. Moreover, the future directions of sample collection and preservation at InBIO are discussed. This development was made possible through the recruitment of the ERA Chair team (WP2), secondments and Junior Researcher exchanges through the collaboration with international networks (WP3), an enhancement of computational infrastructure at InBIO (WP4) and participation of team members in workshops and conferences (WP6). Together, Deliverables D4.2-D4.5 form a detailed account of the successful deployment of a fully functional eDNA lab under the EnvMetaGen project, and provide a valuable resource for eDNA practitioners in all spheres of the triple-helix model.

The overall goal of ERA Chair/EnvMetaGen project No 668981 is to expand the research and innovation potential of InBIO – Research network in Biodiversity and Evolutionary Biology, through the creation of an ERA Chair in Environmental Metagenomics. This field was selected as the focus of the ERA Chair, because Environmental DNA (eDNA) analysis is increasingly being used for biodiversity assessment, diet analysis, detection of rare or invasive species, population genetics and ecosystem functional analysis. In this context, the work plan of EnvMetaGen includes one work package dedicated to the Deployment of an eDNA Lab (WP4), which involves the training of InBIO researchers and technicians for implementing best practice protocols for the analysis of eDNA (Task 4.2). These protocols are essential for key application areas and to the development of research projects in association with business partners and other stakeholders, and thus to the strengthening of InBIO triple-helix initiatives (InBIO-Industry-Government; WP5). This report provides an overview of the current state of the art for the development of best practice for building DNA reference collections of voucher specimens identified by specialised taxonomists, and how these practices are being implemented at InBIO. Building and organising reference collections of DNA sequences is an essential component of this task because eDNA studies require that DNA sequences recovered from the environment are compared to reference collections, which thus need to be developed following consistent and repeatable procedures. Such reference collections are likely to become a tool with significant relevance to the InBIO-Industry-Government triple-helix activities (WP5) by promoting the development of partnerships in all key areas: Monitoring of freshwater eDNA for species detection; Assessing natural pest control using faecal metagenomics; and Next-generation biomonitoring using DNA metabarcoding. Protocols described herein were developed in close connection and due to the accomplishment of the other WPs in the project, including: i) the recruitment of the ERA Chair team (WP2), ii) secondments researcher exchanges through collaborations with international networks (WP3), iii) the enhancement of the computational infrastructure at InBIO (WP4) and iv) participation and organization in workshops and conferences (WP6). Future directions in the improvement to enlarge the reference collection of DNA sequences of InBIO are identified. Together, Deliverables D4.2-D4.5 (this document; Egeter et al., 2018; Galhardo et al., 2018; Paupério et al., 2018) form a detailed account of the successful deployment of a fully functional eDNA lab under the EnvMetaGen project, and provide a valuable resource for eDNA practitioners in all spheres of the triple-helix model.

The overall goal of ERA Chair/EnvMetaGen project No 668981 is to expand the research and innovation potential of InBIO – Research network in Biodiversity and Evolutionary Biology, through the creation of an ERA Chair in Environmental Metagenomics. This field was selected as the focus of the ERA Chair, because Environmental DNA (eDNA) analysis is increasingly being used for biodiversity assessment, diet analysis, detection of rare or invasive species, population genetics and ecosystem functional analysis. In this context, the work plan of EnvMetaGen includes one work package dedicated to the Deployment of an eDNA Lab (WP4), which involves the training of InBIO researchers and technicians for implementing best practice protocols for the analysis of eDNA (Task 4.2). These protocols are essential for key application areas and to the development of research projects in association with business partners and other stakeholders, and thus to the strengthening of InBIO triple-helix initiatives (InBIO-Industry-Government; WP5). This report provides an overview of the current state of the art for collecting and preserving eDNA samples, with particular focus on vertebrate faecal samples, water samples and bulk invertebrate samples, which have been selected as key targets for the development of triple helix strategic initiatives (Task 5.3). The protocols already optimized and currently under development for the collection and preservation of eDNA samples are reported herein. Moreover, the future directions of sample collection and preservation at InBIO are discussed. This development was made possible through the recruitment of the ERA Chair team (WP2), secondments and Junior Researcher exchanges through the collaboration with international networks (WP3), an enhancement of computational infrastructure at InBIO (WP4) and participation of team members in workshops and conferences (WP6). Together, Deliverables D4.2-D4.5 (this document; Ferreira et al. 2018; Galhardo et al. 2018; Paupério et al. 2018) form a detailed account of the successful deployment of a fully functional eDNA lab under the EnvMetaGen project, and provide a valuable resource for eDNA practitioners in all spheres of the triple-helix model. &nbsp

The overall goal of the EnvMetaGen project No 668981 is to expand the research and innovation potential of InBIO – Research network in Biodiversity and Evolutionary Biology, through the creation of an ERA Chair in Environmental Metagenomics. This field was selected as the focus of the ERA Chair, because Environmental DNA (eDNA) analysis is increasingly being used for biodiversity assessment, diet analysis, detection of rare or invasive species, population genetics, and ecosystem functional analysis. In this context, the work plan of EnvMetaGen includes one work package (WP) dedicated to the Deployment of an eDNA Lab (WP4), which involves the training of InBIO researchers and technicians for implementing best practice protocols for the analysis of eDNA (Task 4.2). This report provides an overview of best practices for analysing eDNA samples, focusing on vertebrate faeces, water samples and bulks of invertebrates. It covers all steps from DNA extraction to amplification and sequencing in high throughput platforms, while addressing the challenges identified in each step. Moreover, it describes protocols already optimised and currently under development at InBIO, with particular reference to the detection of single species, diet assessments and biodiversity assessments. These three different outputs are fully aligned with the three key application areas that have been targeted in EnvMetaGen for a wider application of environmental metagenomics, and on which the development of strategic triple helix initiatives will focus on. These will therefore contribute to mainstream this technology as a new cost-effective solution to tackle current challenges faced by business partners and other stakeholders, while fostering InBIO-Industry-Government relationships (WP5 - Strengthening the triple helix). The protocols described herein were developed in close connection to, and build on accomplishments of, the other WPs in the project, including: i) the recruitment of the ERA Chair team (WP2), ii) secondments and researcher exchanges through collaborations with international networks (WP3), iii) the enhancement of the computational infrastructure at InBIO (WP4), and iv) participation in and organization of workshops and conferences (WP6). Future directions in eDNA analyses at InBIO, and other potential laboratory pipelines for future optimisation are identified. Together, Deliverables D4.2-D4.5 (this document; Egeter et al. 2018; Ferreira et al. 2018; Galhardo et al. 2018) form a detailed account of the successful deployment of a fully functional eDNA lab under the EnvMetaGen project, and provide a valuable resource for eDNA practitioners in all spheres of the triple-helix model.

Over the last decade, steady advancements have been made in the use of DNA-based methods for detection of species in a wide range of ecosystems. This progress has culminated in molecular monitoring methods being employed for the detection of several species for enforceable management purposes of endangered, invasive, and illegally harvested species worldwide. However, the routine application of DNA-based methods to monitor whole communities (typically a metabarcoding approach) in order to assess the status of ecosystems continues to be limited. In aquatic ecosystems, the limited use is particularly true for macroinvertebrate communities. As part of the DNAqua-Net consortium, a structured discussion was initiated with the aim to identify potential molecular methods for freshwater macroinvertebrate community assessment and identify important knowledge gaps for their routine application. We focus on three complementary DNA sources that can be metabarcoded: 1) DNA from homogenised samples (bulk DNA), 2) DNA extracted from sample preservative (fixative DNA), and 3) environmental DNA (eDNA) from water or sediment. We provide a brief overview of metabarcoding macroinvertebrate communities from each DNA source and identify challenges for their application to routine monitoring. To advance the utilisation of DNA-based monitoring for macroinvertebrates, we propose an experimental design template for a series of methodological calibration tests. The template compares sources of DNA with the goal of identifying the effects of molecular processing steps on precision and accuracy. Furthermore, the same samples will be morphologically analysed, which will enable the benchmarking of molecular to traditional processing approaches. In doing so we hope to highlight pathways for the development of DNA-based methods for the monitoring of freshwater macroinvertebrates., Metabarcoding and Metagenomics, 3, ISSN:2534-9708

Tese de mestrado em Biologia da Conservação, Universidade de Lisboa, Faculdade de Ciências, 2021 Submitted by Cristina Manessiez (camanessiez@fc.ul.pt) on 2021-05-11T17:22:51Z No. of bitstreams: 1 ulfc126240_tm_Sara_Bento.pdf: 5089616 bytes, checksum: 7ff4992c9b2a3dcf06dfc30dbd308307 (MD5) Made available in DSpace on 2021-05-11T17:23:09Z (GMT). No. of bitstreams: 1 ulfc126240_tm_Sara_Bento.pdf: 5089616 bytes, checksum: 7ff4992c9b2a3dcf06dfc30dbd308307 (MD5) Previous issue date: 2021