NEW YORK (GenomeWeb) – In the midst of the Ebola outbreak in West Africa, researchers with the Liberian Institute for Biomedical Research (LIBR) and the US Army Medical Research Institute of Infectious Diseases (USAMRIID) have set up a genome sequencing laboratory to help manage outbreaks of the virus.
Researchers are currently sequencing Ebola isolates from the outbreak there and plan to use the lab to monitor future outbreaks and emerging pathogens. In addition, along with collaborators from the National Institutes of Health and the Broad Institute, the group has published results of the sequencing of 25 Ebola genomes from patients infected during the outbreak between September 2014 and February 2015 in the journal Emerging Infectious Diseases.
The team chose 25 samples out of a collection of over 1,700 based on the patient's initial diagnostic results indicating a high enough viral load to provide a whole genome. They started with the most recent samples and tested progressively older ones to describe the lineages most likely to still be circulating. They sequenced the 25 samples on five runs of the Illumina MiSeq.
Establishing a genome sequencing laboratory in the middle of a major infectious disease outbreak in an area with limited resources and infrastructure posed a number of challenges, Jeffrey Kugelman, head of bioinformatics at the Center for Genome Sciences at USAMRIID, told GenomeWeb.
The sequencing center was set up at the Liberian Institute of Biomedical Research, which was a well-known and highly regarded research institute in the 1970s prior to the Liberian civil war.
Kugelman said it made sense to base the genome center there because the Liberian National Reference Laboratory was also operating out of the LIBR facility doing diagnostic testing of Ebola samples, and co-locating with the reference laboratory helped to eliminate some redundancies. For instance, the reference lab was already "preparing an inactivated RNA isolate for PCR-based testing, which we could use in the sequencing," Kugelman said.
After finding a space, "the next big issue we ran into was the shipping of the sequencer itself," he said. Traveling halfway around the world is "brutal for a sensitive piece of equipment," he added, noting that even though the MiSeq is relatively "rugged" compared to other next-generation sequencing machines, "there was still significant damage."
In retrospect, Kugelman said having at least two of everything, or at least spare parts for all the commonly replaced components, would have been ideal.
Prior to going to Liberia, Kugelman underwent a two-week training course on the MiSeq — how to install and repair it — since at the time, there were travel restrictions and Illumina personnel would not be able to be on site in Liberia.
So when the machine arrived damaged, Kugelman said he was the one that had to repair it.
The next major consideration was the fact that the lab was located in an area largely devoid of public utilities, like electricity. A generator powered the lab, but the problem with generator power is that there are frequent surges. "The generator just tears through all the electronic equipment." Power transformers and an LED power assembly were two casualties of surges, Kugelman said.
There is also a significant amount of downtime for generators. They can't operate continuously and they have to be shut off to replace the fuel. But, the MiSeq kit is a three-day workflow that needs continuous power, "so we had to add significant backup power supplies," Kugelman said.
Furthermore, average temperatures hovered in the mid to upper 90s and humidity was "outside of the MiSeq's working conditions," so an AC unit had to be installed.
Finally, because the only internet was via satellite, which Kugelman said was slower than dial-up, bioinformatics analysis was done onsite.
Despite the initial challenges, Kugelman said that the LIBR Genome Center is now fully functional and will continue to use NGS to monitor emerging pathogens.
In addition, he said that the recent study and analysis conducted to validate the results shows that the data produced is high quality and that viral genomes can be sequenced within a relevant time frame to make decisions about managing the outbreak.
The lab has a turnaround time for viral genome sequencing of seven days and a throughput of 10 to 20 samples, or about 10 billion bases of sequence data, per week.
For the 25 genomes sequenced in the study, the researchers identified 97 new sequence variants: 47 synonymous, 23 nonsynonymous, 1 nonsense, and 26 noncoding mutations.
Interestingly, the novel nonsense mutation, which was present in two samples, was predicted to cause a premature truncation of an essential protein for viral transcription. However, the authors noted that further analysis will need to be done to determine whether the nonsense mutation or any of the other novel mutations impact fitness of the isolates.
Molecular dating pinpointed the common ancestor of the isolates to between May and July of 2014, corresponding to the early days of the outbreak in Monrovia, Liberia. The viral genomes also indicated that there was a single introduction event in Liberia, and then diversification within the country. All 25 samples likely originated from the same haplotype thought to have been circulating in Sierra Leone and Guinea in late May 2014.
The team also identified a handful of mutations that could potentially impact drug sensitivity; however, they were at a low frequency and none of the samples had more than one change per drug type.
Suzanne Mate, a researcher with USAMRIID that worked with the LIBR Genome Center to train local scientists to use the MiSeq, said that the next steps are to finish analyzing the data. In addition, she said that LIBR will continue to run the MiSeq in other infectious disease and outbreak scenarios. Now that the lab is set up and operational, sequencing and analysis could potentially be used to make clinical management decisions in future cases, she said.