How do you stop outbreaks becoming epidemics in real time?

Birmingham researchers placed genomic sequencing at the heart of the Ebola and Zika outbreak response.

It is a time of change for those at the cutting edge of genome sequencing. Professor Nick Loman, at the University of Birmingham, is very aware of the speed of change.

“Two decades ago, the publication of the first bacterial genome sequence, from Haemophilus influenzae, shook the world of bacteriology. Since then we’ve seen a complete transformation of our understanding of how bacteria function, evolve and interact with each other, with their hosts, and with their surroundings. Now we are able to sequence the entire genetic code of a disease-causing microbe or pathogen. The third decade promises to be just as exciting.”

Since that first genome sequence, three ‘revolutions’ have shaped the direction of progress: whole-genome shotgun sequencing, high-throughput sequencing and single-molecule long-read sequencing.

The powerful combination of genome sequencing and bioinformatics-driven analysis of sequence data has seen projects that used to take years and cost hundreds of thousands of dollars now being completed in a few days for less than the price of a meal out for two.

“I fully expect the gold rush to continue and to see the $1,000 human genome matched by the $1 bacterial genome. I think we’ll see a 'sequencing singularity', whereby sequencing becomes the method of choice for as-yet unthinkable applications. They have already managed the encoding and then sequencing Shakespeare's sonnets in a DNA format, there are so many directions this could take.”

Taking the lab on the road

Indeed, the benefit of the advances is already being felt in very real terms.

In April 2015, Professor Loman’s PhD student, Josh Quick, arrived in Conakry, Guinea.

The Ebola outbreak in West Africa was the deadliest occurrence of the disease since its discovery in 1976. Since the first confirmed case was recorded on 23 March 2014, over 11,000 people died as the result of the virus. Despite a coordinated international response to the outbreak, it proved extremely difficult to control.

The ambition was to deploy a portable “laboratory in a suitcase”, allowing for Ebola samples from patients to be sequenced as soon as new cases were diagnosed. With less than 50kg of luggage, and thanks to a novel DNA sequencer, the team were able to do just that.

“Genome sequencing information is crucial for researchers and epidemiologists during an epidemic. Yet, generating such information is a laborious process typically performed in well-equipped laboratories using large, delicate and expensive hardware. Having a portable DNA sequencing system opens up the possibility to do outbreak genome sequencing in real-time, which can directly impact on the response on the ground, as well as providing a wealth of information about pathogen evolution.”

The team found that they could generate sequencing information in as little as 24 hours after receiving a sample, with the sequencing process taking less than an hour.

By comparing samples from patients, it was possible to determine whether they were likely to be part of a chain of transmission or if there were unknown networks yet to be discovered. This works because the virus mutated at a constant rate – about 20 differences per year. By comparing the number of differences between samples, it is possible to predict whether they form part of a recent transmission chain or network.

That is useful in understanding the evolution of the virus and aiding control efforts – but only if it can be rapidly provided given to epidemiologists battling to halt transmission of the virus.

“That is where the ‘lab in a suitcase’ comes in. There was not a lot in Josh’s pack - three laptops, some chemical reagents, a centrifuge and a thermocycler we ‘borrowed’ from a lab mate. But the crucial component was a lightweight DNA sequencer called a MinION, made by Oxford Nanopore Technologies. This allowed us to create a fully functioning laboratory that could collate and provide genome sequence information in real time. Crucially, the team working to combat Ebola was on the same page – and by sharing our data and making it available as it was generated we could help provide the information that would shape the development of vaccines.”

Tackling a new challenge

The team barely had time to take stock after the success of the genomic surveillance of the Ebola outbreak before shifting their focus towards Brazil, and the spread of Zika.

“Zika was spreading across the Americas and the Pacific and geneticists were playing catch-up. There were very few publicly available DNA sequences and hardly any from the regions where cases of microcephaly are most prevalent.”

So, using the same model for mobile laboratories that the team had deployed in Guinea, the team took to the roads of north-east Brazil to detect and characterise the early emergence of Zika in large urban centres. Over a 30-day sampling period the team acquired samples from patients in coastal Brazil - from Belém itself in the north to Salvador in the east.

The study showed that Zika’s establishment within Brazil - and its spread from there to other regions - occurred before Zika transmission in the Americas was first discovered.

“We found that northeast Brazil, which was the region with the most recorded cases of Zika and microcephaly, was the nexus of the epidemic in Brazil and played a key role in its spread within Brazil to major urban centres, such as Rio de Janeiro and São Paulo, before spreading across the Americas. Now we have a much better understanding of the epidemiology of the virus.”

“It just goes to highlight the importance of creating trusted cross-institutional partnerships and sharing data openly during disease focused research field work. By not being precious about your data and embracing a more open way of working we can expediate the understanding of the outbreak, and in doing so help public health officials to get it under control and, ultimately, save lives.”

This new method of in-situ genomic surveillance, paired with open data, the team developed a protocol which can be beneficial to other researchers working in remote areas around the world during times of viral outbreaks.

Before the headlines

It has been quite a journey for the Loman group. Though the work on Ebola and Zika made global headlines, their history in genomic surveillance can be traced back further.

In 2011, the team analysed genome data from a German outbreak of Escherichia coli, one of the first to have genome data made available while it was taking place. They were able to show that the strain causing disease was of a type previously unseen in such outbreaks, and tracked the likely source to a strain circulating in humans, rather than animals.

Then in 2014 the team, using the MinION for the first time, were able to successfully study a Salmonella outbreak at a hospital in the West Midlands.

These studies show that foodborne outbreaks can be detected back to source much more quickly and, in the future, food quality may be monitored by producers at source using genomics – all thanks to portable genome surveillance.

“The nanopore technology has changed the field, without a doubt. Rather than waiting for outbreak samples to arrive at sequencing facilities, it allows us why not take the facility to the outbreak?”

A nanopore is exactly what you would think: a small hole.

Most commonly it consists of a small peg-shaped protein with a hollow tube at its core, just a few billionths of a metre wide. In the Oxford Nanopore device used by the Loman lab, the protein sits in a synthetic membrane, submerged in liquid.

When a voltage is applied, creating an electric current across the membrane, ions start to flow through the hollow tube. However, when something blocks the hole, such as a strand of DNA, the ions are impeded and the current drops.

Somewhat handily, the four bases of DNA - A, C, G, and T— change the current in different ways. Ergo if you measure the current, you can decipher the sequence of a DNA strand as it threads through the pore.

What the future holds

The key to the future of this research might well have been signalled by a paper published earlier this year. Using the same MinION technology, the team were able to sequence the most complete human genome ever assembled with a single technology.

At 1,204,840 bases in length, it was 8,000 times longer than a typical sequencing read and more than a thousand times longer than the original reads used to generate the human genome reference sequence in 2001.

As well as sequencing previously uncharacterised regions of the genome, the new analysis provided greater insight into regions of the genome that are responsible for functions such as immunity and tumour growth. This in turn could have a profound impact on clinical practice, for example, detecting large genome rearrangements important in the development of cancer and in determining a person’s inherited repertoire or antibody genes.

Being able to sequence using a portable device that only costs £1,000 may put also personalised genome sequencing into the mainstream.

“Until even just a year ago, it would have been impractically difficult to sequence a whole human genome, but thanks these recent advances and innovations we are making that evermore possible. If you imagine the process of assembling a genome together is like piecing together a jigsaw puzzle, the ability to produce extremely long sequencing reads is like finding very large pieces of the puzzle which makes the process far less complex.”

The inevitable challenge has arrived though. With technology now allowing for far quicker sequencing, the infrastructure is needed to be able to analyse the data at the same rate.

That is why Birmingham is part of CLIMB – the Cloud Infrastructure for Microbial Genomics - a multi-million pound project that is investing in bioinformatics and building ‘big data’ capacity for the UK genomics community.

The heart of the project is four sites; Birmingham, Warwick, Cardiff and Swansea, with a connected virtual computing infrastructure, optimised for specific applications such as microbial genome alignment, de novo assembly and metagenomics.

“The speed of change is really something. As our technology and infrastructure continue to make great leaps, the possibilities for researchers grow exponentially. When you think of where we were in 2001, with a multi-billion, decades-long project to sequence the human genome, and where we are now with sequencing labs on the International Space Station, we know how far we have come. And yet there is so much more on the horizon.”

The work mentioned in this article was only possible with numerous collaborators including:

Ebola

The European Mobile Laboratories, Public Health England and Ontario Institute for Cancer Research, other diagnostic laboratories and WHO epidemiologists based in Guinea.

Zika

Instituto Evandro Chagas, the Institute of Tropical Medicine, University of São Paulo and the Fundação Oswaldo Cruz (FIOCRUZ), in Salvador, Bahia, Medical Research Council (MRC), Public Health England, the Universities of Oxford, Nottingham and Edinburgh, as well as the University of Sydney, Australia and the Ontario Institute for Cancer Research, in Toronto, Canada. The project was funded by the Medical Research Council’s Zika Rapid Response Initiative, USAID, and supported by the Wellcome Trust and the Newton Fund.

Sequencing human genome

Universities of Nottingham, East Anglia, California, Salt Lake City, British Columbia and Toronto, as well as NIH’s National Human Genome Research Institute.

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