• Research

Ship-seq: Nanopore sequencing of polar microbes on board icebreakers

Using Oxford Nanopore Technologies MinION for in situ, real-time sequencing of polar ocean microbes.

Project summary.

Led by: Leggett Group

Start date: Oct 2017

End date: May 2021

Duration: 4 years

Grants: NERC NEXUSS DTP studentship

We are working on using Oxford Nanopore Technologies MinION for in situ, real-time sequencing of polar ocean microbes. Polar oceans are some of the most underexplored ecosystems in the world, with relatively little information about the microbial species which live there, despite their huge diversity and importance. Microbes, such as phytoplankton, which live in polar oceans are important in carbon capture, food-webs, and biogeochemical cycles of elements such as Nitrogen, Iron, and Silica.

Polar ecosystems are under significant threat from climate change and we don’t yet have a good understanding of how phytoplankton and other microbes will be affected, or what knock-on effects this will have. To understand this, and to create models for further research, we need to develop a better picture of which species are present where, and understand more about their life cycles, interactions, and responses to changing conditions. We are undertaking metagenomic DNA sequencing of polar ocean samples to achieve this.

DNA sequencing samples from polar oceans has been difficult because it takes months for the samples to get back to the lab, with unquantifiable loss of diversity occurring during storage. This is particularly problematic with metagenomic samples, where differences in culturability and DNA degradation rates can introduce biases to the species found. To counter this problem, we are using Oxford Nanopore MinION sequencing in situ, removing the need for sample storage and cell culturing prior to sequencing.

We will also perform real-time analysis to determine which species are present, using NanoOK. Real time information on which species are present could allow researchers to make evidence-based decisions on where and when to sample, saving vital resources.

Details.

Psychrophilic phytoplankton such as diatoms are particularly dominant in polar oceans, due largely to their genetic adaptability which has allowed them to successfully and rapidly develop strategies for survival in extreme conditions. Relatively little is known about the genetic basis for this adaptability, and it is important that we improve our understanding of this if we are to model the impacts of climate change effectively.

Currently there are very few genome assemblies available, which limits research in this area. The lack of genome assemblies is partly a result of the inaccessibility of their habitat, and partly because their genomes are often complicated with heterozygosity, repeating regions, and unpredictable ploidy.

The Oxford Nanopore Technologies MinION is a small (10 x 3 x 2 cm, 90 g), portable DNA sequencing machine. It is powered by a USB connection and can be run on a laptop computer. The sequencing works by feeding a DNA strand through a biological pore one base at a time, identifying bases by their ionic current.

This continuous strand sequencing means that in theory an entire DNA strand could be sequenced. There are 2048 pores in the MinION flow cell, allowing for significant coverage to be generated in one run. This method has advantages over short-read shotgun technologies such as Illumina, as long-reads allow for improved de novo genome assembly and provide more information on genome structure.

Long read metagenomic MinION sequencing of polar ocean samples would allow us to produce assemblies of a wide variety of polar ocean microbes. This would facilitate investigations into the genetic basis of the incredible adaptability of diatoms. The library preparation technique requires only a small number of reagents and can be carried out without extensive laboratory equipment.

In situ sequencing prevents the loss of information caused by long-term sample storage and real-time base-calling, in conjunction with NanoOK for real-time analysis, means that within 48 hours of taking a sample we can determine which species are present.

Collaborators.

Thomas Mock

School of Environmental Sciences, UEA

Vincent Moulton

School of Computing Sciences, UEA

Clara Manno

British Antarctic Survey

Impact statement.

This is a proof-of-concept study to show that probing and analysing polar microbes at the ocean-atmosphere interface can be done while on an expedition in polar oceans. In situ real-time MinION sequencing of metagenomic polar phytoplankton samples could solve many of the problems currently faced by researchers in this field.

The portability and lack of equipment requirements for sample preparation allow immediate sequencing of samples, thus preventing degradation and community alteration. The rapidity of MinION sequencing allows for the use of DNA sequencing data to be used in determining the progress of a research cruise, based on the identification of species of interest.

Long-read sequencing raises the possibility of using this data to build a reference database of polar phytoplankton which would aid research into genetic adaptations to changing conditions, improving our understanding of their response to climate change.