Developing methods for long-read marine viral metagenomics

Everybody knows how important terrestrial plants are to the global climate, fixing atmospheric carbon and providing the oxygen that sustains life. What is less known is that marine microbes are responsible for around half of total global primary production. Seawater is teeming with microbes, with around 5 million in every teaspoon in surface water. These can be categorised as the phototrophs that fix atmospheric carbon, the heterotrophs that convert this carbon back to atmospheric CO2 and the vast number of viruses that interact with these two groups. At any given time, around 30% of marine microbes are infected by their associated viruses. Viral lysis kills 20% of bacterial standing stocks every day, releasing a soup of dissolved organic carbon that is the largest pool of available carbon on Earth. Heterotrophs rapidly convert this back into CO2, preventing it from sinking to the deep where it is locked away. Viruses can also act as agents of gene transfer, and can reconfigure host metabolism during infection, changing their metabolic inputs and outputs. Such changes then resonate throughout the community, ultimately shaping its function and composition.

Traditionally, our understanding of viruses was limited to those for which we had successfully cultured the host. As >99% of microbes are uncultivated, this provided a very narrow representation of viral diversity. Recent advances in sequencing viruses directly from the environment, by preparing viral metagenomes (viromes) have revolutionised our understanding of the importance, abundance and impact of viruses on global biogeochemical cycles. However, such approaches rely on successfully assembling viral genomes from short read data, similar to reconstructing an enormous genomic jigsaw with billions of pieces. The high rates of evolution and extraordinary diversity of viruses makes this a challenge. Currently <1% of viral sequences from a metagenome can be assigned to a known viral group.

This project will establish new methods to sequence viral metagenomes using the latest long-read sequencing technology. PacBio and Nanopore sequencers generate reads that are several thousand basepairs long, in some cases, sufficiently long to span an entire viral genome. This increased length makes assembly and analysis of viral genomes trivial. To date, application of long-read sequencing to viral metagenomics has been limited by a need for DNA input requirements that were orders of magnitude higher than that which can be reasonably extracted from an environmental viral sample. However, recent updates to library preparation for Nanopore and advances in DNA amplification for PacBio make it an opportune time to re-evaluate their use in viral metagenomics.

As a result of long-read sequencing, extracting full length viral genomes from environmental samples will provide a step-change in the resolution with which we can perform viral ecology.

The development of methods to sequence viral communities with significantly greater fidelity offers broad benefits to many applications. Products of this research will have implications for a wide range of stakeholders interested in the use of this tool on fisheries assessments, aquaculture pathogen detection, conservation biology, environmental risk management (e.g. toxic algae blooms, human pathogens, ballast water regulations), with the wider aim of supporting biodiversity and nature's services through NERC's strategic pillar of "Managing environmental change" and the EU's Marine Strategy Framework Directive (MSFD) Good Environmental Status (GES) key Biodiversity Maintenance descriptor 1.

As part of the London Calling Nanopore event, we will
1. Showcase the Nanopore as a tool for screening of aquatic viruses with a live demo of newly developed protocols
2. Engage with water monitoring bodies (e.g. Environment Agency) and the aquaculture industry to develop in-situ monitors for viral and bacterial threats

Under the banner of the SeA-DNA project (NE/N006100/1), which this proposal supports, we will also ensure that developed methods are presented at a proposed stakeholder workshop aiming to:

1) evaluating the needs of the stakeholder community; and
2) determining an individualised roadmap of engagement with each stakeholder group.

Exploiting strong institutional relationships and strategic alliances within the SeA-DNA project, we plan to engage: DEFRA (MSFD implementation); CEFAS (fisheries assessments); IMO Joint Group of Experts on the Scientific Aspects of Marine Environmental Protection - GESAMP (ballast water); Census of Marine Life (Biodiversity); European Centre for Environment and Human Health (human pathogens); British Ecological Society (conservation biology); and the Marine Management Organisation - MMO (marine sustainability and policy).

The project specifically addresses the GES Descriptor: 4 (Elements of food webs ensure long-term abundance and reproduction). Application of viral sequencing tools, including amplification of low concentration DNA and in-situ sequencing with Nanopore, could advance MSFD implementation.