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Nanopore Sequencing Becomes More Portable to Enable Outbreak Surveillance in Field

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SAN FRANCISCO (GenomeWeb) – Ever since Joshua Quick boarded a plane for Guinea three years ago with all the equipment needed for sequencing Ebola patient samples contained in one checked suitcase, researchers have been looking to develop even more portable methods to sequence infectious disease samples outside of a traditional laboratory.

Quick, at the time a graduate student in Nick Loman's lab at the University of Birmingham in the UK, brought his equipment to the European Mobile Laboratory Project during the Ebola outbreak in Guinea in 2015, where more than two dozen Ebola samples were sequenced. The sequence data was used to identify transmission patterns that were then communicated to public health officials.

Since then, a number of research groups have developed protocols for field-based infectious disease testing with nanopore sequencing, including a team from MRIGlobal that is working on a nanopore sequencing-based "lab in a backpack."

Researchers from Public Health England in the UK have focused on developing a metagenomic sequencing protocol that is applicable to any RNA virus and have already conducted nanopore sequencing in the field to inform outbreak management. Meanwhile, researchers from the University of Göttingen in Germany have focused on speed and portability — developing a so-called suitcase lab that can sequence samples in less than seven hours with minimal equipment and no internet connection.

Loman's team at the University of Birmingham, meantime, has continued to develop sequencing, library prep, bioinformatics, and phylogenetics methods under a grant from the Wellcome Trust Collaborative. That work, which the team has made available online, has focused on speeding up the process and making it cheaper and more robust, Loman said in an email.

The Public Health England team, which had worked with Quick on the Ebola sequencing project in West Africa, wanted to build on the portability of that approach, but with a metagenomic method, as opposed to one that targeted Ebola, in order to be able to detect any RNA virus in a sample.

In July, the researchers described in a preprint a metagenomic sequencing protocol for RNA viruses directly from clinical samples, without nucleic acid amplification or enrichment, and have already used it to help inform an outbreak of Lassa fever in Nigeria.

Lassa virus is endemic in Nigeria and there are typically seasonal outbreaks in the country, similar to influenza in the US and Europe.

Liana Kafetzopoulou, a doctoral student at Public Health England who spearheaded the work in Nigeria, along with collaborators from the Bernhard Nocht Institute for Tropical Medicine in Hamburg, Germany, said that "the plan was to go and do a pilot study during one of the endemic seasons." However, "during the course of the endemic season, it became apparent that Nigeria was facing the largest ever recorded increase of Lassa fever cases."

As such, the pilot study quickly turned into a real-time outbreak surveillance project that was used to inform public health officials. The group had set up at the Institute of Lassa Fever Research and Control in Nigeria, which also housed a diagnostic lab. PHE sequenced samples that had already been diagnosed and found to be positive, a total of 120 patient samples that were analyzed on the MinIon between February and the middle of March of this year.

As soon as sequencing results were available, the researchers fed that information back to the Nigeria Centre for Disease Control and the World Health Organization and made them available online.

One of the main goals of the project was to determine how the virus was being spread, Kafetzopoulou said. Lassa virus is carried by rodents and is not known to be transmissible between people.

"But, there was an unprecedented number of cases compared to previous years," Kafetzopoulou said, "so we really wanted to know whether there was human-to-human transmission or the emergence of a novel strain." Sequencing was able to confirm that this was fortunately not the case. "Because the viruses weren't closely related, we ended up being able to conclude that it was independent transmission from rodent to human rather than human-to-human transmission," she said.

Understanding the transmission pattern was critical for planning how to manage the outbreak. Because transmission was from the natural host, the public health focus was on avoiding contact with rodents. If, however, the virus had been found to be spreading from person to person, the response "would have focused on tracing all persons who had contact with a Lassa fever patient," Kafetzopoulou said.

Since the initial work, the team has continued to build capacity in Nigeria and to work with scientists there to use sequencing for outbreak surveillance for Lassa and other diseases. She said the main application would be to inform outbreak management rather than to provide diagnoses. One benefit of the nanopore approach was that they were able to set up in a laboratory with limited resources, at the hospital where patients were being diagnosed, rather than in a centralized reference laboratory far removed from the patients, she said.

Being able to test in limited-resource settings is an advantage of the MinIon's small size, but Kafetzopoulou noted that one challenge was that there were frequent power outages in Nigeria. In addition, data analysis relies on an internet connection.

A group from the University of Göttingen has been focused on developing a protocol that would address the power and lack of Internet issue, with a solar-powered suitcase lab that can do offline data analysis.

Originally, the team's setup relied on isothermal amplification rather than sequencing to detect Ebola virus. However, the group "realized that almost 40 percent of the samples are always negative," said Ahmed Abd El Wahed, a University of Göttingen researcher. "We don't know if that's because the samples are truly negative or because we're not detecting the pathogen." Thus, the group switched over to metagenomic sequencing on the MinIon and recently described its approach in the Journal of Clinical Virology.

"The idea was to establish a protocol where we can take the sample and do metagenomic sequencing directly," which would allow for agnostic analysis rather than targeting a specific microbe or set of microbes, Abd El Wahed said.

In the recent study, the group described its metagenomic sequencing approach, which involves multiple displacement isothermal amplification for sample prep followed by sequencing and an offline bioinformatics analysis. As a proof of concept, they demonstrated the protocol on a mock sample that contained Zika virus.

The team extracted RNA and performed reverse transcription, which also eliminated genomic DNA, and then used the NEBBext mrRNA kit for sample prep.

Abd El Wahed said that one key for sequencing in the field was to ensure that all the reagents and equipment could survive without having to be refrigerated or kept on ice. The reagents the researchers chose could be used at 25°C for one day, but for longer-term storage, a freezer would still be needed.

After sequencing on the MinIon, the researchers used a custom-designed offline bioinformatics process. Typically, nanopore sequencing data is analyzedin the cloud. But that is not feasible in the field, Abd El Wahed said. Previously, groups have done the sequencing portion in the field, but the analysis had to be done either in a lab or at a hotel with an internet connection. But because the team wanted to do the bioinformatics at the site of sampling and sequencing, it developed an offline method.

"We established an offline genome search with a viral genome library," he said.  The library included more than 7,000 viral genome assemblies that were available in NCBI databases.

The entire process — including sample prep, sequencing and analysis — took less than seven hours and generated more than 63,000 reads. Zika virus sequences were identified in around 4 percent of the reads.

Abd El Wahed said that currently, the main application is in outbreak investigations, not diagnostics. The method is "quick and dirty," he said, with no purification or depletion steps, and the goal isn't to get a full genome but to find out what's there quickly. Most of the process involves sample prep, with just 20 minutes of sequencing time and under an hour of offline data analysis.

"The main application is rapid detection of an outbreak," he said.

One ongoing challenge is that the shelf life of reagents and the MinIon flow cells is relatively short, about two months, Abd El Wahed said. Ideally, he'd like to see the shelf life be extended to around one year.

Going forward, he said, he plans to make the protocol more robust. His team has ongoing collaborations with the Institute Pasteur de Dakar in Senegal to evaluate the method for detecting arboviruses and with Makerere University in Uganda for investigating animal diseases. In addition, the team is working with researchers at the University of Stirling in Scotland to develop offline data analysis tools.

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