Bio-On-Magnetic-Beads (BOMB): Open platform for high-throughput nucleic acid extraction and manipulation

Current molecular biology laboratories rely heavily on the purification and manipulation of nucleic acids. Yet, commonly used centrifuge- and column-based protocols require specialised equipment, often use toxic reagents and are not economically scalable or practical to use in a high-throughput manner. Although it has been known for some time that magnetic beads can provide an elegant answer to these issues, the development of open-source protocols based on beads has been limited. In this article, we provide step-by-step instructions for an easy synthesis of functionalised magnetic beads, and detailed protocols for their use in the high-throughput purification of plasmids, genomic DNA and total RNA from different sources, as well as environmental TNA and PCR amplicons. We also provide a bead-based protocol for bisulfite conversion, and size selection of DNA and RNA fragments. Comparison to other methods highlights the capability, versatility and extreme cost-effectiveness of using magnetic beads. These open source protocols and the associated webpage (https://bomb.bio) can serve as a platform for further protocol customisation and community engagement

Global monitoring of antimicrobial resistance based on metagenomics analyses of urban sewage

Antimicrobial resistance (AMR) is a serious threat to global public health, but obtaining representative data on AMR for healthy human populations is difficult. Here, we use meta-genomic analysis of untreated sewage to characterize the bacterial resistome from 79 sites in 60 countries. We find systematic differences in abundance and diversity of AMR genes between Europe/North-America/Oceania and Africa/Asia/South-America. Antimicrobial use data and bacterial taxonomy only explains a minor part of the AMR variation that we observe. We find no evidence for cross-selection between antimicrobial classes, or for effect of air travel between sites. However, AMR gene abundance strongly correlates with socio-economic, health and environmental factors, which we use to predict AMR gene abundances in all countries in the world. Our findings suggest that global AMR gene diversity and abundance vary by region, and that improving sanitation and health could potentially limit the global burden of AMR. We propose metagenomic analysis of sewage as an ethically acceptable and economically feasible approach for continuous global surveillance and prediction of AMR.Peer reviewe

Setting a baseline for global urban virome surveillance in sewage

The rapid development of megacities, and their growing connectedness across the world is becoming a distinct driver for emerging disease outbreaks. Early detection of unusual disease emergence and spread should therefore include such cities as part of risk-based surveillance. A catch-all metagenomic sequencing approach of urban sewage could potentially provide an unbiased insight into the dynamics of viral pathogens circulating in a community irrespective of access to care, a potential which already has been proven for the surveillance of poliovirus. Here, we present a detailed characterization of sewage viromes from a snapshot of 81 high density urban areas across the globe, including in-depth assessment of potential biases, as a proof of concept for catch-all viral pathogen surveillance. We show the ability to detect a wide range of viruses and geographical and seasonal differences for specific viral groups. Our findings offer a cross-sectional baseline for further research in viral surveillance from urban sewage samples and place previous studies in a global perspective

Soil environmental DNA metabarcoding in low-biomass regions requires protocol optimization: a case study in Antarctica

Environmental DNA is a powerful tool for monitoring biodiversity. Although environmental DNA surveys have successfully been implemented in various environments, protocol choice has been shown to affect results and inferences. Thus far, few method comparison studies for soil have been undertaken. Here, we optimized the workflow for soil metabarcoding through a comparative study encompassing variation in sampling strategy (individual and combined samples), DNA extraction (PowerSoil®, NucleoSpin® Soil, PowerSoil® + phosphate buffer and NucleoSpin® Soil + phosphate buffer) and library preparation (one-step and two-step quantitative polymerase chain reaction methods). Using a partial 18S rRNA marker, a total of 309 eukaryotic taxa across 21 phyla were identified from Antarctic soil from one site in the Larsemann Hills. Our optimized workflow was effective with no notable reduction in data quality for a considerable increase in time and cost efficiency. The NucleoSpin® Soil + phosphate buffer was the best-performing extraction method. Compared to similar studies in other regions, we obtained low taxonomic coverage, perhaps because of the paucity of Antarctic terrestrial organisms in genetic reference databases. Our findings provide useful methodological insights for maximizing efficiency in soil metabarcoding studies in Antarctica and other low-biomass environments

Environmental DNA (eDNA) metabarcoding surveys show evidence of non-indigenous freshwater species invasion to new parts of Eastern Europe

Active environmental DNA (eDNA) surveillance through species-specific amplification has shown increased sensitivity in the detection of non-indigenous species (NIS) compared to traditional approaches. When many NIS are of interest, however, active surveillance decreases in cost- and time-efficiency. Passive surveillance through eDNA metabarcoding takes advantage of the complex DNA signal in environmental samples and facilitates the simultaneous detection of multiple species. While passive eDNA surveillance has previously detected NIS, comparative studies are essential to determine the ability of eDNA metabarcoding to accurately describe the range of invasion for multiple NIS versus alternative approaches. Here, we surveyed twelve sites, covering nine rivers across Belarus for NIS with three different techniques, i.e. an ichthyological, hydrobiological and eDNA survey, whereby DNA was extracted from 500 ml surface water samples and amplified with two 16S rDNA primer assays targeting the fish and macroinvertebrate biodiversity. Nine non-indigenous fish and ten non-indigenous benthic macroinvertebrates were detected by traditional surveys, while seven NIS eDNA signals were picked up, including four fish, one aquatic and two benthic macroinvertebrates. Passive eDNA surveillance extended the range of invasion further north for two invasive fish and identified a new NIS for Belarus, the freshwater jellyfish Craspedacusta sowerbii. False-negative detections for the eDNA survey might be attributed to: (i) preferential amplification of aquatic over benthic macroinvertebrates from surface water samples and (ii) an incomplete reference database. The evidence provided in this study recommends the implementation of both molecular-based and traditional approaches to maximise the probability of early detection of non-native organisms

Beyond Biodiversity: Can Environmental DNA (eDNA) Cut It as a Population Genetics Tool?

Population genetic data underpin many studies of behavioral, ecological, and evolutionary processes in wild populations and contribute to effective conservation management. However, collecting genetic samples can be challenging when working with endangered, invasive, or cryptic species. Environmental DNA (eDNA) offers a way to sample genetic material non-invasively without requiring visual observation. While eDNA has been trialed extensively as a biodiversity and biosecurity monitoring tool with a strong taxonomic focus, it has yet to be fully explored as a means for obtaining population genetic information. Here, we review current research that employs eDNA approaches for the study of populations. We outline challenges facing eDNA-based population genetic methodologies, and suggest avenues of research for future developments. We advocate that with further optimizations, this emergent field holds great potential as part of the population genetics toolkit

Non-invasive real-time genomic monitoring of the critically endangered kākāpō

We used non-invasive real-time genomic approaches to monitor one of the last surviving populations of the critically endangered kākāpō (Strigops habroptilus). We first established an environmental DNA metabarcoding protocol to identify the distribution of kākāpō and other vertebrate species in a highly localized manner using soil samples. Harnessing real-time nanopore sequencing and the high-quality kākāpō reference genome, we then extracted species-specific DNA from soil. We combined long read-based haplotype phasing with known individual genomic variation in the kākāpō population to identify the presence of individuals, and confirmed these genomically informed predictions through detailed metadata on kākāpō distributions. This study shows that individual identification is feasible through nanopore sequencing of environmental DNA, with important implications for future efforts in the application of genomics to the conservation of rare species, potentially expanding the application of real-time environmental DNA research from monitoring species distribution to inferring fitness parameters such as genomic diversity and inbreeding

Assessing the utility of marine filter feeders for environmental DNA (eDNA) biodiversity monitoring

Aquatic environmental DNA (eDNA) surveys are transforming how marine ecosystems are monitored. The time-consuming preprocessing step of active filtration, however, remains a bottleneck. Hence, new approaches that eliminate the need for active filtration are required. Filter-feeding invertebrates have been proven to collect eDNA, but side-by-side comparative studies to investigate the similarity between aquatic and filter-feeder eDNA signals are essential. Here, we investigated the differences among four eDNA sources (water; bivalve gill-tissue; sponges; and ethanol in which filter-feeding organisms were stored) along a vertically stratified transect in Doubtful Sound, New Zealand using three metabarcoding primer sets targeting fish and vertebrates. Combined, eDNA sources detected 59 vertebrates, while concurrent diver surveys observed eight fish species. There were no significant differences in alpha and beta diversity between water and sponge eDNA and both sources were highly correlated. Vertebrate eDNA was successfully extracted from the ethanol in which sponges were stored, although a reduced number of species were detected. Bivalve gill-tissue dissections, on the other hand, failed to reliably detect eDNA. Overall, our results show that vertebrate eDNA signals obtained from water samples and marine sponges are highly concordant. The strong similarity in eDNA signals demonstrates the potential of marine sponges as an additional tool for eDNA-based marine biodiversity surveys, by enabling the incorporation of larger sample numbers in eDNA surveys, reducing plastic waste, simplifying sample collection, and as a cost-efficient alternative. However, we note the importance to not detrimentally impact marine communities by, for example, nonlethal subsampling, specimen cloning, or using bycatch specimens