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Emma Langan - Ship-Seq: nanopore sequencing of polar microbes onboard research vessels

London Calling 2019

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.

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Emma Langan began her presentation describing her PhD project, which she began in October 2017, spending a year “learning the ways of the nanopore” and trying to extract DNA from phytoplankton. The major part of the presentation would cover Emma’s trip taking the MinION to see what she could do with polar microbes on board a research cruise vessel.

Emma explained that her core interest is in ocean phytoplankton, which are responsible for 50% of primary production across the globe, as well as being responsible for carbon cycling. In particular, a subset called diatoms have silica shells, meaning they are slightly heavier than other plankton and so sink to the bottom of the ocean when they die, taking carbon with them instead of releasing it back into the ecosystem.

Diatoms exist primarily in polar oceans, and they are very adaptable, preferring to live in niches that are not yet occupied by other species. Emma also described them as “very cosmopolitan”, possessing much more diversity between species than you might expect. Polar phytoplankton are the basis of polar food webs, and many organisms, up to large mammals such as polar bears, rely on their existence to survive.  Polar phytoplankton also contribute disproportionately to primary production, producing a huge amount of chlorophyll across all seasons.

Despite their importance in the Earth’s ecosystem, very little is known about phytoplankton, with only two genomes having been sequenced and assembled – one alga and one diatom – and previous studies have not covered polar oceans.

Bearing this in mind, Emma posed her research questions: are populations of polar phytoplankton changing with climate change? Can we use genetics, instead of traditional microscopy, to identify which species are present in different locations? And what do polar phytoplankton have that allows them to exist in such extreme environments where other species can’t?

Emma aimed to work towards answering some of these questions on her research cruise, trying to capture species which can’t be cultured or stored to be brought back to institutions for further analyses.

Taking a look at her methods, Emma detailed the reasons why they chose to use MinION to investigate these questions. Some species of phytoplankton simply don’t survive in culture or in storage, and DNA degrades over time, so on-site analysis was a must. In addition, via other methods it can take months to get data back to work with; Emma described how another group on board her vessel in January are still yet to have actionable data in late May. By contrast, using MinION allowed Emma to have data to work with within hours of beginning sequencing. This meant data could be used to make informed decisions; for example if they needed to return to a sampling site if the sample contained species that need further investigation.

Finally, Emma explained that short reads do not return good metagenomic assemblies, and if their project was to try and increase the number of phytoplankton genomes in reference then long reads would be essential. Summarising, Emma showed a picture of herself on board the Discovery in February, saying “Take your MinION, a couple of computers, and you’re good to go!”.

Moving on to the results of her study so far, Emma described how the team took samples at 12 stations, and she went on to sequence 3 of those samples whilst still on board the ship. Moving past “statutory penguin photos”, she detailed how a CTD was used to obtain 100 litres of water per station, as to obtain a microgram of DNA requires the filtration of tens of litres of water. In fact, this step was a real bottleneck for Emma and the team, as it took some ten hours to filter all the water they required.

DNA extraction was performed with phenol chloroform, which is incredibly toxic, but works effectively to remove DNA from tough polar phytoplankton. The sequencing library preparation was done with the ligation 109 kit, before analysis with NanoOK-RT, a real time version of Richard Leggett’s algorithm that has been used everywhere from this expedition to characterising the microbiome of preterm infants.

The average read length obtained was 1 kb, which, considering the best obtained read length in a lab in optimal conditions was 10 kb, was a good achievement in-field on a moving ship. There was enough sample to do later confirmatory sequencing, and 4.63 Gb data was generated across the sequencing runs. Emma mentioned this was “nothing special”, but the circuitous route to the Falklands may have impacted reagents and performance!

Highlighting some examples in the results, Emma showed their detection of Emiliana huxleyi in their northernmost sample, a microbe that had been reported as encroaching on the Southern ocean – potentially confirming that report. Detection of Phaeocystis was also exciting to Emma and the team, as it smells like sulphur, so they knew it was there before seeing its appearance in the sequence data! Emma also found some diatoms; lots of different species in low amounts, so not enough to assemble, but enough to confirm their highly diverse presence in the water.

Concluding her presentation, Emma explained the limitations of her work, most prominently taking dangerous phenol chloroform aboard a ship (“not health and safety ideal!”), and the high proportion of unclassified organisms in the study reflecting the lack of sequenced genomes in databases.

The future direction of the project is to continue confirmatory sequencing and do further analysis using metadata from the ship, for example temperature or nutrient level information, and going on additional research cruises to continue sampling. Summarising, Emma stated that phytoplankton are important, and polar phytoplankton even more so. We don’t know much about phytoplankton, and even less about polar phytoplankton, so there is much to be done. MinION should be a good way to find out more, coupled with better extraction methods and better reference databases.

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