NEW YORK (GenomeWeb) – In a study appearing online today in Nature, an international team shared findings from an Ebola virus sequencing study done in West Africa using its portable, real-time nanopore sequencing lab.
As GenomeWeb reported last June, researchers from the European Mobile Laboratory Consortium and elsewhere transported three Oxford Nanopore Technologies MinIon instruments, along with four laptop computers, a PCR thermocycler, a heat block, reagents, and consumables, to West Africa last April.
"Since then, really, we've scaled it up … to try to capture as much of the outbreak as possible," co-first author Nicholas Loman, a microbiology and infection researcher at the University of Birmingham, told GenomeWeb.
At the original lab at Donka Hospital in Conakry, Guinea or at the Coyah Ebola Treatment Unit, where the equipment subsequently moved, the researchers sequenced the genomes of 142 Ebola virus isolates collected in Guinea between March and October last year.
By comparing these genomes to one another and to Ebola viruses sequenced by other teams during the West African outbreak, they identified two main Ebola virus lineages behind cases in the region over much of last year.
Rather than directly sequencing the Ebola virus' single-stranded RNA, which is often contaminated with human transcripts that can swamp out viral sequences, the group used targeted RT-PCR to isolate and amplify genetic material from the virus.
The researchers initially came up with a set of 38 primer pairs to produce amplicons prior to sequencing, though they whittled this down to 11 long amplicons after a few days in the field. In samples where they ran into amplification problems that produced missing genome regions, the protocol was modified to focus on 19 shorter amplicons per sample.
The team passed the resulting genome sequences on to the World Health Organization and to National Coordination in Guinea.
For their own analyses, the researchers considered all but 17 low quality samples, combined with 603 previously sequenced Ebola virus isolates. As in past studies, they saw substitution rates that resembled those described for the Ebola virus prior to the outbreak.
This stable evolutionary rate makes it possible to use mutations that have cropped up since the West African outbreak began about two years ago to define distinct lineages that have emerged in the interim, Loman explained.
In particular, the Guinean samples his team considered fell into two main lineages. One of these, the GN1 lineage, is closely related to Ebola viruses found in Guéckédou, Guinea very early on in the outbreak. Since then, the GN1 lineage has mainly been found in Guinea and, to a lesser extent, in Sierra Leone.
The other lineage, known as SL3, has been mainly linked to cases in Sierra Leone, though it appears to have made its way to the Conakry, Guinea region by late 2014.
By combining the MinIon genomes with Ebola virus sequences generated by researchers in Sierra Leone, the team estimated that the GN1 and SL3 lineages have taken separate migration paths leading to Ebola virus infections in Guinea and Sierra Leone in 2015.
"We're essentially looking at two separate migration paths for the virus," Loman said. "One probably coming through Guinea — from east Guinea up through the more urban areas … around Conakry in western Guinea — and another lineage that got into Sierra Leone, spread in Sierra Leone, and then [traveled] up the coast to western Guinea."
Though the West African outbreak is now largely contained, Loman noted that the team is prepared to sequence any additional cases that flare up. "In Liberia and Sierra Leone, even after the outbreak was declared over, we've seen reemergence of cases," he said. "Fortunately, we haven't seen any in Guinea."
The researchers have set up a third portable, real-time sequencing location in Nongo, Guinea and are continuing to look for ways to optimize their protocols to boost speed, cut back on sample sizes, and perhaps reduce reliance on internet connectivity for base calling.
Beyond the insights it offers into Ebola virus dynamics during 2015, Loman called the new Nature paper a "how-to guide" for those working on other types of outbreaks in the field, particularly in a resource-limited setting.
While his team does not plan to tackle the Zika virus themselves, for example, he noted that the portable sequencing strategy might benefit groups planning to use genomics to study those infections in resource-limited locations.