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Shaili Johri - Unraveling shark secrets: sequencing genomes and microbiomes for research and conservation

London Calling 2019

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. 

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Shaili opened her talk by boldly asking: why save sharks and rays? We are typically fearful of them (cue an image of Jaws!). Yet according to Shaili, sharks can be "adorable" and "they are gentle giants... generally". 

Jokes aside, Shaili stated that sharks are crucial for the functioning of healthy oceanic ecosystems; they keep mesopredators in check, which is important for maintaining the oceanic food chain at a healthy equilibrium. They are also major contributors to the economy. Sharks have "been around for a really long time, so they are very evolutionarily resilient"; in fact, "everything about sharks is really long!" For example, they have a long life history, a long development time... and therefore require extraordinary genome stability to protect them from a potentially large accumulation of mutations over a long lifetime. 

Despite their importance, up to 50% of shark species are threatened by extinction, due to the international demand for shark fins and other shark parts or derivatives. We are "fishing them out", with ~300 million sharks killed by humans per year. To put this into perspective, there have only been 20 shark attacks on humans in last decade - they should be far more scared of us than we are of them.

Conservation of endangered populations and an understanding of their evolutionary adaptations are both difficult when genomic information is lacking for over 50% of shark species. Globally, the areas where sharks are most endangered also correlates with the areas of highest data deficiency; i.e. we don't know enough about the sharks in these locations. Shaili suggested that this makes it easier to fish for them and exploit them.

The goal of Shaili and her team has been to reduce the data deficiency of shark populations by performing on-site genomic investigations in shark biodiversity hotspots. We need to figure out the population sizes of the species present, including the presence of interbreeding subpopulations; we need to determine the distribution of species and what is threatening them. A lot of species identification is typically based on Cytochrome oxidase I gene amplicon sequencing, which doesn't differentiate between species, or sometimes the gene simply does not amplify. This information therefore does not help advise us in determining the best strategy for species conservation and management. Shaili and her team wanted to come up with a method that could be used by anyone, anywhere to help inform best practices for shark species conservation and management.

Shaili took her research to India - the second largest global shark supplier. Shark meat is often seen in fish markets in India, typically from a range of species; as the shark fins are often separated from the body, the species can't be identified, and so whether the meat is being sold legally cannot be determined. In her research, samples were obtained from shark fin specimens; genomic DNA was then extracted and sequenced using the Oxford Nanopore MinION platform. Shaili applied a "genome skimming" shallow whole-genome sequencing approach using a single MinION flow cell per sample. High copy number mitochondrial sequences were taken and used for taxonomic identification and phylogenetic analysis, and high copy number nuclear sequences were used to study population size and structure. With in-field genome skimming, Shaili has achieved 99.8% sequence accuracy, coverage of a sixth of the shark genome, sequence lengths of up to 100 kbp, and very high GC percentage coverage. One problem that she faced was that contigs aligning to the mitogenome appeared to be longer than the mitochondrial genome. She discussed that these reads were probably coming from a replicating mitochondria whose DNA hadn't been cut yet in the cell, "so that was an easy fix".

With nanopore sequencing, fast and accurate species identification was performed, most confidently within 3 hours, but they "could start determining species ID within 2 minutes". She compared this timeline to Sanger sequencing which would typically take about 24 hours to achieve a similar result. By integrating sequence data acquired in the field with local databases through the Geneious software, which has a "super easy GUI interface" that does not require any bioinformatics, Shaili's data has also contributed to an increased capacity for wildlife forensics. For example, the silky shark species was identified in the fish market - trade of this species is illegal as it is protected. Confirmation of her in-field results was performed back in the lab using Canu alignment. With the presence of higher computing power compared to out in the field, more sequence contigs were also obtained and other genes were covered, such as homeobox genes, and genes involved in immune system function and genome stability. This was particularly exciting, as these genes had not been identified before.

Another interesting observation was that the GC percentage of the shark genomes ranged from 29.5-60%. This linked to how important sequencing versatility is in wildlife forensics, as the genomes of endangered species have a range of GC content; for example, elephant genomes have ~39% GC content, sharks and rays ~42% GC content, and rhinos ~51% GC content. Therefore, the methods that Shaili has used for wildlife forensics could be applied to the conservation of a range of species because they have obtained such good efficiency across a range of GC% in sharks.

Next Shaili briefly described how her team has identified that a lot of fish and chip shops in the UK often sell shark meat, although this is generally unknown to the shop owners. The method could therefore also be used to detect shark meat in fish and chip shop food.

Lastly, Shaili wanted to focus on the monitoring of shark populations. She stated that it is important to monitor microbial microfauna, as this is important for shark health and environmental monitoring. Therefore, in addition to her investigations into shark population genomics and conservation, Shaili has also been investigating the microbiomes of free-swimming whale sharks (Rhincodon typus) across the globe, in order to identify threats such as disease, pollution and habitat degradation. In practical terms: "how do we sample sharks"? Most often, free-swimming sharks are sampled underwater; this is performed using a device that seals on to the shark skin, which flushes microbes off the shark skin to isolate them. She declared that the main issue with this process is keeping up with a swimming shark! They have not yet tried swimming next to thresher sharks; those sharks which they cannot swim alongside are brought on board for sampling. 

This project has also contributed to the research training of undergraduates in the lab. Students have been directly involved in sampling, DNA extraction, and nanopore sequencing and analysis, to investigate the taxonomy and function of the microbiomes detected. One of the key findings that has been made is that the genomes are rich in heavy metal metabolising genes; they are currently investigating why this might be.

Shaili concluded that her team will continue to work with species identification and the sequencing of shark genomes, as well as wildlife forensics, and lastly, she will continue to study population genetics using genome skimming data. This research has demonstrated how the portable sequencing technology of Oxford Nanopore has improved the genetic understanding of shark populations, which will ultimately facilitate the protection of endangered shark species, and potentially other endangered wildlife species.

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