MinION Below Zero: sequencing microbes and marine mammals, birds, and the Alaskan coastal environment from low-quality samples, including whale snot

Microbial interfaces: within hosts, between hosts, and the environment

Eric Bortz kicked off his talk with an overview of his lab’s work sequencing the microbial interface of the Alaskan coastal environment. Eric described how Alaska is on the edge of a changing Arctic with complex ecological networks, multiple species interfaces, and vast untapped genomic reservoirs, which are poorly understood. Eric highlighted the average temperature has increased 3 to 4 degrees in northern latitudes, and sea-ice levels are at their lowest since the 1960s, characterising the impact of climate change is having in this region.

Eric described other drivers of the changing environment, including human-impacted ecology, pollutants, and radioactive contamination, all of which are stressors for various species within the ecosystem.

Lab research overview

The emergence of wildlife pathogens is a strong focus of the projects undertaken at the Bortz Lab; work to date has identified RNA viruses, bacteria (Coxiella) and eukaryotic parasites successfully using nanopore sequencing. Examples included SARS-CoV-2, influenza, and TB in humans, and coronaviruses, influenza, paramyxoviruses, African swine fever, and bacteria in animals.

Searching for avian and marine mammal pathogens in the coastal waters of Alaska, Eric highlighted the challenges of obtaining high-quality samples, as most are obtained from stranded animals and dead birds, resulting in low-quality genetic or microbial material.

Eric detailed that one of his previous graduate students, Amy Klink, developed methods for sequencing microbial organisms, particularly viruses, from these ‘hard to get environmental samples, from species such as stranded marine mammals’. Additionally, another student, Maile Branson who has been working on avian influenza, developed a workflow for reverse transcription multiple displacement amplification (RT-MDA) for random amplification of RNA to increase the amount of RNA available for sequencing.

Eric described how this approach was cost-effective for labs like his. In this setting, Eric demonstrated proof of principle on cancer cells (A549 cell lines), which they use to study respiratory viruses, indicating 25x amplification of a small amount of DNA diluted from these samples.

Eric then presented his teams work applying this RT-MDA technique to northern fulmar lung tissue, with sequencing on the MinION. Of 305,102 samples, 221,168 were classifiable, of which 45,880 were bacterial, and 1,310 were viral. Analysis of data with Kraken2 and Pavian identified a range of pathogens including influenza A virus.

A description of how avian influenza can spread to other animals, including the whales from birds, was given, followed by details of the eight genome segments which make up the influenza genome. To sequence these genomes, the Ligation Sequencing Kit (LSK109) performed the best, followed by reference-based assembly with minimap2 as part of an iterative process which, in turn, allowed for virus phylogenies to be inferred.

Using this same method again on stranded marine samples (seals, whales, sea lions, and sea otters), amplification and sequencing with nanopore technology revealed avian flu in the braincase of a beaked whale.

Live great whale blow sampling by drone collection

Ocean Alliance (Adam Rogan), U Alaska (Shannon Atkinson), and colleagues brought Eric into the project using drones to sample and sequence whale blow samples. The technology was tested off Stellwagen Bank, MA; Baja CS Mexico; Southeast Alaska. As part of this work, Eric analysed the whale blow using multiple displacement amplification with sequencing on the MinION, obtaining read lengths with N50 19900, enabling him to characterise the samples.

Respiratory metabiome of the humpback whale

Taxa identified in seawater contained bacteria, eukaryotes (likely planktonic plants and fungi), archaea, and viruses (bacteriophages and marine ssDNA viruses).

Whale unique gram-negative aerobic bacteria of genus Paenalcaligenes and Vitreoscilla were also identified in these samples. Taken together, the results presented show how the research undertaken amplified low-quantity and low-quality nucleic acids and used nanopore sequencing to understand emerging RNA viruses in avian and marine mammal species in Alaska.

Conclusion

To conclude the talk, Eric described the use of nanopore sequencing to understand emerging RNA viruses in avian and marine mammal species in Alaska. Steps taken were amplification of low-quantity and low-quality nucleic acids, which led to identifying a novel avian influenza-like virus in Stejneger’s beaked whale.