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Cheryl L. Ames - Field-forward sequencing with Oxford Nanopore technology: a strategy to establish the upside-down mangrove jellyfish Cassiopea xamachana as a bioindicator

London Calling 2019

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.

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Cheryl Ames (US Naval Research Laboratory) kicked off the aquatic ecosystems panel plenary with a reminder of the importance of the ocean, "the source of every breath on Earth." Oceans cover 72% of the Earth's surface and are a home to most of the Earth's biodiversity; however, this is under threat from climate change, activities such as over-fishing, and natural disasters. Cheryl stressed the need to study the ocean's biodiversity "before it's too late."  She presented the work of her team using eDNA to categorise this diversity, for environmental monitoring, sting prevention (affecting both combat divers in the Navy and recreational divers), to investigate biodiversity in the gulfstream, and also aid public aquariums.

Cheryl described how she and her team decided to focus on jellyfish eDNA, which has multiple sources: from spawn, gametes and stinging mucus. She showed the upside-down jellyfish (Cassiopea xamachana) in mangrove forests, releasing spawn and mucus, and the spawning of the venomous box jellyfish (Alatina galata) in coastal waters; both events provide good sources of eDNA and enable the detection of these organisms even if the visible jellyfish are not seen or are no longer present in the area. The team selected these two jellyfish for further study, as they are emerging model organisms; they are about to publish the genomes for both, together with a third genome, adding to the very few jellyfish assemblies currently available for use as references genomes. Cheryl also noted that only one other paper currently discusses the use of jellyfish eDNA. Showing the different stages of the life cycle of jellyfish, from microscopic to macroscopic, Cheryl further demonstrated how much more there was to jellyfish than is visible to the naked eye.

Cheryl then highlighted how she and her team work on developing tools to ensure the safety of service members, whilst being "conscious and conscientious of the environment", in the locations they visit around the globe. Quoting its versatility and portability, she described how "the MinION was really the answer to what we wanted to do." She showed that the team had taken advantage of the portability of the MinION device to conduct sequencing experiments in multiple locations, from on a cliff-face to in a car ("all rental cars should come with a MinION, in case you want to sequence at the airport"). The MinION was implemented in their investigation of the effect of natural disasters on populations of jellyfish. Cheryl showed Buttonwood Sound, Key largo in the Florida Keys: the bay emptied in the aftermath of Hurricane Irma, and with it disappeared the Cassiopea jellyfish populations. She noted that even eight months after the emptying of the bay, only a few Cassiopea could be found.

To sequence jellyfish eDNA, sampling was performed at 7 collection sites: 2 protected from the hurricane, 4 exposed to the hurricane and 1 positive control from an aquarium. Water was filtered, the eDNA extracted and pooled. 16S amplification was performed and the samples sequenced in multiplex on the MinION, run from a battery. Metabarcoding and bioinformatic analysis were performed using guppy basecalling, Porechop and Cutadapt. The NCBI 16S database and proxy sequences and QIIME2 were then used, though Cheryl noted that the latter was designed for short-read microbiome data; Cheryl displayed the full eDNA metabarcoding analysis workflow, featuring taxonomic classification, diversity analysis and phylogenetic analysis. The results demonstrated rarefied alpha biodiversity, with 50 observed taxonomic units representing ~50 species of jellyfish in 4 classes. She noted that she wasn't surprised to detect species in protected areas ("we saw plenty of them there"), but jellyfish species were also detected in exposed oceanic areas, where they had not been visible. Phylogenetic analysis of the Florida Keys jellyfish again showed species representing all four classes; focusing on the Cassiopea taxa, Cheryl showed that the visibly-seen C. xamachana was abundant, but C. andromeda, which had not been spotted, was also detected. The positive control involved sampling of an aquarium housing a C. frondosa jellyfish, and this species was successfully identified; it was also identified at the Buttonwood Sound site much more than at the other five. Then focusing on the box jellyfish taxa, Cheryl showed that several species were represented quite well; she pointed out that the venomous box jellyfish was identified in sites used for diving training where they were not visibly seen - important data for avoiding stings.

Cheryl concluded that field-forward sequencing of eDNA with the MinION, operated via battery, enabled the successful detection of organisms. She highlighted that the method is "approaching real-time assessment" in even austere environments and could be modified to suit any system.

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