Applying portable nanopore sequencing technology to the conservation of the critically endangered kākāpō

"I really enjoy being out here...being on an island with kākāpō, even when you don't see them all the time...the feeling is really great."

Lara came to her current role, looking at how to apply statistical genomics and other molecular approaches to support nature conservation, as she has "always been interested in the ecological as well as the computational and bioinformatic research directions". From a background in ecology and bioinformatics which she studied for her undergraduate degrees, to a PhD at the University of Cambridge focused on statistical cancer genomics, she has brought all of her experience together into her current role in conservation genomics. Lara told me that biodiversity and nature conservation have always been her passion, and were the reasons for her initially studying biology at university. But Lara is also very interested in how we can make use of "big data" – a necessity to study, and preserve, something as complex as nature. For her PhD she decided to really "dig deep and learn statistical genomics properly", and it was from there that she could follow her passion and start to apply all of that knowledge to conservation issues.

Every species plays its role in an ecosystem

To Lara, the kākāpō is "a very special animal". It was actually during her PhD in the cancer field that this interest arose, having read about the critically endangered kākāpō, of which, at that time, there were less than 200 individuals left in the entire world. She was aware of a genomics project that had been started to sequence the entire species; this was the first time that every individual of a species would be sequenced, which seemed like "a cool milestone". Having contacted the lead scientist in the program, after her PhD Lara obtained a Humboldt fellowship from the German state and moved to work together with conservationists in New Zealand. "They are just really fantastic animals" Lara reflected, explaining how she got to see and hold a kākāpō for the first time. Whilst reminding us that we shouldn't just focus on conserving these iconic species, Lara did highlight, however, that this example is very representative of what we might lose if we don't start preserving biodiversity right now: "We know that every species plays its role in an ecosystem so it does make sense to try and preserve every species – or at least to make sure that it's not us as the human species that brings them to the brink of extinction". Moreover, in the case of the kākāpō, these animals have taken over the role that large grazing mammals tend to have in the rest of world, such as seed dispersal, placing them as a central species in the New Zealand ecosystem.

Genomic information is central to species conservation

Lara explained how "a lot more is happening on the genomic level than what we actually see just in the phenotype of individuals". We don't understand what is going on at the genomic level if we just look at phenotypes, particularly where there is a high level of inbreeding; there may be specific molecular pathways that lead to an increased disease susceptibility or decreased reproductive success, for example. So, to understand mechanistically the "genomic architecture" of the traits that might be important for a species' recovery, genome information is really important, and something that Lara focuses on in her research. Genomic information also allows you to understand just how inbred, or how in danger, a species is, in order to organise effective global conservation efforts. Just counting individuals doesn't give an insight into genetic relatedness and how a species might be able to adapt to future changes.

Portable, real-time nanopore sequencing technology has been fundamental to protecting the kākāpō

The decision to use nanopore sequencing was based on the in situ and real-time aspects that have been key to Lara's work.

"Being able to get results really quickly has really convinced me that this is the way forward to any sort of point-of-care genomic approaches in the future."

Moreover, as there was no initial high investment, Lara saw this as really important to for making the technology "accessible for everyone". She began using nanopore sequencing during her PhD at the University of Cambridge, where, with a few friends and colleagues, she started a project alongside her PhD to use nanopore sequencing to assess the microbial load of the River Cam. Even with little funding, and needing to learn how to do the lab-side of things, which Lara admitted isn't her strong point as a computational biologist ("that's actually another advantage of nanopore sequencing, that it's...very easy to learn, even back then in 2017"), she and her colleagues showed that it was possible with the portable MinION sequencer to "in a very reliable, robust manner" determine the pathogenic composition of the river. You can check out her PuntSeq blog and eLife publication on that research.

When Lara moved to New Zealand it became increasingly clear to her that the technology would be fundamental to her work; especially in New Zealand, where there is sovereignty of the indigenous Māori people over data associated with their "taonga" (treasured) species and their "whakapapa" (genealogy), the ability to locally produce and store genomic data facilitates consultations and adherence to ethical guidelines. Being able to create high-quality reference genomes in situ with the portable MinION sequencer was therefore ideal – and is something of high importance in Lara's recent work too, where she has been contributing to the genome assembly of endemic dolphin species together with her New Zealand collaborators (via the initiative). Furthermore, it is "very important to get information from the genomic data directly in the field" to adjust kākāpō conservation management decisions. She is currently using portable nanopore sequencing to protect the kākāpō population from another Aspergillosis outbreak that has impacted the species during the latest breeding season. Lara explained that, traditionally, when you would get samples from the kākāpō chicks to check for a fungal infection, the samples would be sent out the next time a helicopter arrived on the island – which might be up to two weeks. "In the end, I guess what I want to say is, the chicks will be dead long before we receive any genomic results if there is actually an Aspergillosis outbreak".

Lara also drew my attention to a collaboration of her colleague Jo Stanton with researchers in Africa to monitor Cassava virus outbreaks, and thereby improve harvest for local farmers there; and "that's the same story – if you have to send off your sample to get results then by the time you have those results, you will have lost significant amounts of your harvest. For us, it is further very important to empower local communities and researchers to use and benefit from genomic approaches on their own".

"A very special memory that I have from one of my fieldwork trips is when I actually got to hold my first kākāpō. It was an individual of old age, and he seemed to be falling asleep on my lap. What is so amazing about kākāpō, besides their particular appearance, is their odour – they smell like the forest they live in. Before releasing it, we gave it an almond, which they really enjoy, and released it – that was a fantastic experience, followed by many more amazing kākāpō encounters."

Real-time, targeted nanopore sequencing (adaptive sampling) has great potential for widespread, non-invasive environmental monitoring

Lara has recently started using adaptive sampling, a method for real-time, on-device target enrichment or depletion, on the GridION, "quite extensively, because it very easily allows you to predominantly sequence the genomic data that you are interested in". She explained how sequencing environmental DNA (eDNA) from soil samples, using real-time adaptive sampling, enabled her to select for and sequence kākāpō DNA, allowing them to genomically monitor kākāpō in a completely non-invasive manner. Going beyond the sequencing of "iconic species", eDNA will be a vital source of genomic data – Lara hopes that in the future eDNA could be used to study the genomics of all co-occurring species in an environment at the same time.

Long reads are crucial for assembling high-quality reference genomes

The provision of long reads is another aspect of nanopore sequencing technology that has benefited Lara's work, alongside its portability and real-time capabilities. Firstly, "when it comes to reference genome creation, they are basically crucial to create a high-quality reference genome", as only long reads allow you to "put the pieces where they should belong". In general, Lara stated, the longer the read, the more useful it is. For example, regarding the kākāpō eDNA-based research, long reads enabled her to assign haplotypes, and it was only by having these haplotypes that she could then identify individual kākāpō – just from environmental material. As kākāpō are so inbred, small "snippets of DNA cannot resolve haplotypes successfully" because they might map to any kākāpō individual's genome.

Accessible genomics: bringing the technology to the local community

We moved on to further discuss how Lara has been working with the local communities on the kākāpō recovery project. With the kākāpō recovery team, from the national Department of Conservation, Lara is now benchmarking all her approaches, so, when they are optimised, the team will have all the knowledge and technology to be able to apply it themselves. As the laboratory work is "not so difficult" ("even I can easily do it as a computational biologist!"), she is pretty confident that nanopore sequencing could be taught to everyone.

Lara has also been an advisor of the Accessible Genomics Project, which aims to bring nanopore sequencing to the global South, especially in the light of the COVID-19 pandemic. She stressed that what is important is to not take the science away from local communities or financially disadvantaged countries in order to then publish that research in as high an impact journal as possible, but to actually bring the science there and "leave it there, so people can use it on their own in the long term".

"Our goal is to actually establish the technology with stakeholders, such as local communities or conservationists, so that they can use it on their own in the future."

The work is physically and mentally challenging – but always very rewarding in the end

Fieldwork can be challenging, Lara stated, especially if you do it for quite a long time; it can be both physically and mentally tough. But it is also one aspect of her work that she always really looks forward to. The challenging part is often optimising everything in the laboratory – to do things that haven't been done before, combining new and different approaches, getting one negative result after another, and then finally bringing everything into the field, with literally everything packed into a single backpack. "This, paired with the general pressure that scientists are under to accomplish tasks as fast as possible", and with many other obligations alongside, "has often been a mental challenge", but it is "always very rewarding in the end". "I did have my moments when I thought it would not work, but we got there – you always get there at some point".

Everything needed for fieldwork fits into a single hiking backpack

For sequencing in the field, Lara's team uses the Rapid Sequencing Kit, "which is easily deployable in the field". So all you need is the kit, the nanopore sequencer, including the Flow Cell Priming Kit, and then some beads and a magnetic rack if you want to do a bead clean-up. That is all easily portable. When it comes to the DNA extraction, they use a BentoLab; and while this machine is a little bigger and heavier than the other reagents, it is still portable and it can run on a battery. They also have their portable embedded mini-computers with powerful GPUs for in-field analysis (these have been obtained via a collaboration with Miles Benton at ESR). All of this fits into a single hiking backpack along with the other essential provisions.

Holistic visions for the future

In New Zealand, Lara has been focusing on two endangered flightless birds, the kākāpō and the takahē. Lara wants to now "take the next step" and understand more broadly how genomic approaches can be used to contribute to the enhancement of planetary health: "the concept that all health, of all systems on earth, is connected", i.e. that the health of nature is connected with human health as well. Lara emphasised the importance of understanding how our own health depends on the health of nature and the environment. This is something that Lara really wants to explore further – how big data and molecular biology intersect with human health.

Lara also gave the specific example of studying bioaerosols and how they impact human health – be it monitoring air in hospitals or airports, to detect dangerous pathogens leading to infections, and to prevent any future pandemics.

"Going more into this holistic direction of using genomics, especially portable real-time analysis that will directly tell us what's going on, to understand the world around us, is something that I would like to drive forward as much as I can with the help of many other nanopore scientists out there".

This research is a collaboration with the Kākāpō and Takahē Recovery Teams from the New Zealand Department of Conservation, ESR Wellington, the Kākāpō Aspergillosis Research Consortium, University of Otago, and the Alexander von Humboldt Foundation.

Want to learn more?

Watch Lara's plenary talk on "Leveraging adaptive sampling of environmental DNA for monitoring the critically endangered kākāpō" at NCM 2021 Discover how nanopore sequencing is being used to support sequencing of critically endangered species

View our white paper on "New insights into large genomes"

Explore animal genomics research with nanopore technology

Urban, L. et al. Freshwater monitoring by nanopore sequencing. eLife. 10:e61504 (2021).