Field-forward sequencing with Oxford Nanopore technology: a strategy to establish the upside-down mangrove jellyfish Cassiopea xamachana as a bioindicator
The combined relative ease of operation, high throughput and reduced cost of NGS platforms has enabled the coupling of traditional field collection methods with laboratory-based metagenomic approaches to provide a molecular snapshot of species-diversity in a plethora of aquatic environments. However, the often-extensive time-lag between field sampling, sequencing and endpoint bioinformatics analyses precludes the ability to provide a contemporaneous characterization of the target ecosystem, against the backdrop of briskly shifting global climate. Furthermore, the growing decline of healthy aquatic ecosystems due to chemical, physical and biological threats, along with concerns related to invasive species and natural disasters, highlights the critical need for field-forward sequencing protocols to provide rapid characterization of a diversity of environmental systems. The recent publication of the reference genome of the upside-down mangrove jellyfish, Cassiopea xamachana, has gained this emerging model species attention as an indicator species with promising applications for coastal ecosystem management and conservation. Taking advantage of the versatility offered by Oxford Nanopore sequencing, we developed a field-forward DNA environmental metabarcoding strategy to characterize Florida Keys mangrove ecosystems, inhabited by C. xamachana, in the year following the catastrophic landfall of Hurricane Irma in 2017. The prototype for this portable system boasts a low-complexity protocol requiring minimal training for operation, a relatively short sample-to-answer timeframe i.e. several hours, field-forward DNA metabarcoding capabilities in austere environments, manual and/or battery-powered equipment with ease of portability and minimal footprint, as well as multiplexing capabilities for the simultaneous assessment of multiple collection sites and/or genetic markers. We present here the first ever eDNA assessment of C. xamachana populations in several Florida Key coastal environments in the wake of a devastating natural disaster, based on the findings of our inaugural field-forward sequencing study.
Cheryl L. Ames is a National Academies of Sciences (NRC) Postdoctoral Fellow at the United States Naval Research Laboratory and a Research Associate at the National Museum of Natural History, Smithsonian Institution in Washington, DC. As an expert in jellyfish and their evolutionary novelties, such as their venom, vision and sex, Cheryl uses a MinION-based field-forward environmental DNA sequencing protocol to assess biodiversity in marine ecosystems, such as coastal mangroves, the Gulf Stream and public aquaria. Cheryl has a Ph.D. in Biological Sciences from the University of Maryland, USA, a Master’s degree in Marine Biology from the University of the Ryukyus in Okinawa, Japan and undergraduate degrees from Queen’s University and the University of Ottawa in Canada.
Ship-Seq: nanopore sequencing of polar microbes onboard research vessels
One of the exciting features of the MinION is the ability to carry out in situ sequencing in remote environments where previously it has been impossible to effectively study ecosystems. Polar oceans are biodiversity hot-spots which disproportionately contribute to global biogeochemical cycles, but they are among the most under-explored ecosystems on Earth, as well as the most threatened by anthropogenic environmental change. As a result of this, there is increasing interest in the study of polar microorganisms such as diatoms and coccolithophores, which are the main regulators of the polar ocean biogeochemical cycles. The study of polar microbes is often challenging as they survive only at specific temperatures, which limits our ability to transport them to laboratories for experiments. Long-term maintenance in the laboratory is also problematic as many species are cold-adapted and require polar-specific environments, as well as failing to thrive in close quarters. We are addressing this challenge by using the MinION for real-time studies on the diversity and function of microbial communities from the surface ocean. Our aim is to provide a real-time assessment of microbial diversity, real-time analysis of in situ experiments in polar oceans, and genome and transcriptome sequencing of sensitive but ecologically relevant polar microbes. During January and February 2019, we carried out our first feasibility test during a research cruise on the RRS Discovery, in which a MinION was used in conjunction with NanoOK RT software for in situ sequencing and real-time analysis of metagenomic samples collected by the ship. Our experiment provided information about species composition and abundance at multiple sampling points on a long transect between the Falkland Islands and South Georgia and the South Sandwich Islands, crossing the polar front. This includes a range of nutrient levels and temperatures, which allows for the investigation of genetic basis for the ability of diatoms and other phytoplankton to survive in a wide variety of conditions. Real-time analysis onboard a research vessel allows researchers to make evidence-based decisions on sampling locations and whether sampling has been sufficient. The results from our experiment will be validated against previous data from similar locations, alongside sequencing of sample replicates using alternative platforms. This will allow for a comparison between in situ sequencing with the MinION and UK-based sequencing with other platforms. Our results indicate that MinION sequencing is a powerful tool for polar microbe research, although a lack of available reference genomes currently limits its power. For further investigations, alongside the production of more reference genomes, analysis pipelines will be tailored to target specific genes and species that are of interest in terms of their function and ecological role.
Emma Langan is a NEXUSS PhD student at the University of East Anglia, Earlham Institute and the British Antarctic Survey, where she is using the Nanopore MinION for real-time metagenomic sequencing of polar ocean samples to monitor microbe populations. Emma graduated from University of Edinburgh with a BSc in Biomedical Sciences (Infectious Diseases) in 2015, before completing a MSc in Bioinformatics where she built a genome browser for the investigation of silent cricket genotypes.
Unraveling shark secrets: sequencing genomes and microbiomes for research and conservation
Chondrichthyes - sharks, rays and chimaeras (‘sharks’) evolved 500 million years ago and are one of the oldest extant vertebrates today. Sharks have extraordinarily long-life spans, exceptional wound healing capabilities and large genomes – qualities which make them ideal candidates for understanding mechanisms contributing to genome stability and immunological resilience. As apex predators, sharks are also vital to top-down regulation of oceanic ecosystems and are therefore crucial to maintaining commercial fish stocks and human food security. However, sharks are disproportionately targeted to meet the international demand for shark fins and as a result an estimated 25-50% of species are threatened by extinction. 50% of shark species are also data deficient, making it difficult to conserve remaining populations and to study their evolutionary adaptations. Our goal is to reduce data deficiency of shark populations through on-site genomic and metagenomic studies in shark biodiversity hot-spots, including the USA, India, Tanzania, Mexico, Australia, and Philippines. Shark samples are collected from free swimming sharks or from specimens found in fish markets. Genomic DNA is sequenced on-site by trained undergraduate and graduate students on the MinION. We sequenced four new chondrichthyan genomes including the Silky shark (Carcharhinus falciformis), Sharpnose guitarfish (Glaucostegus granulatus), and two manta rays (Mobula japonica and Mobula tarapacana). Long-read sequencing on the MinION allowed high depth of sequencing coverage of shark genomes, which are typically 1-6 gigabases in size. Our studies increased the number of sequenced chondrichthyan genomes by 40%. Ongoing genome assessments for population size and structure will allow determination of conservation status for these shark species. Genome comparisons across taxa will increase understanding of mechanisms which impart evolutionary resilience to this species group. Further, our microbiome analyses of free-swimming whale sharks (Rhincodon typus) in locations across the globe revealed that microbiomes are similar with respect to taxonomic composition and functional profiles in genetically diverse and geographically separated whale shark populations, providing key insights about the biogeography of whale sharks. Analyses of functional profiles of the microbiome in wild thresher sharks (Alopias vulpinus) revealed a10-fold higher proportion of heavy metal-metabolizing genes in sharks as compared to the water column in coastal San Diego, suggesting either bioaccumulation of heavy metals or a novel baseline microbiome specific to thresher sharks. In summary, use of portable sequencing technology from Oxford Nanopore has improved the data deficiency of shark populations through local capacity building and will facilitate greater protection of endangered species in the future.
Shaili Johri is a geneticist with a research focus in conservation genomics of wildlife populations. She completed her BSc. and M.S. in India and moved to the United States for her PhD in Genetics. She did her post-doctorate at the Center for Conservation Biology at the University of Washington in Seattle and is currently a research professor at San Diego State University in association with Dr. Elizabeth Dinsdale’s laboratory. Shaili works at the intersection of marine conservation policy and interdisciplinary research and her research career spans projects relating to conservation of tigers in western India, wolves in northwest USA, killer whales in the Pacific Northwest and now sharks and rays in the southwest US and India. Shaili’s role as a project lead involves developing genomic and metagenomic tools to assist with biodiversity assessments and population health monitoring of marine megafauna such as sharks and killer whales. In parallel to her research, Shaili works in close collaboration with fishing communities to develop science-based conservation policies through cross-sector collaborations, outreach and education. In addition to her research, Shaili engages in capacity building for genomic methods among wildlife research communities in the US and abroad and this is where the Oxford Nanopore MinION device has been a game changer.