May 2015

Next-Generation Sequencing Aids Researchers in
the Fight Against the Ebola Virus
Genomic studies with the HiSeq® and MiSeq® Systems are enabling researchers to track Ebola
outbreaks and understand the impact of the virus’s rapid mutation rate.
Introduction
The 2014 Ebola outbreak sickened and killed thousands of people
in West Africa, struck major urban centers and sent countries intochaos, and caused an international and humanitarian crisis. But first,
the virus infected a child. Through careful history taking, researchers
identified a two-year old boy as the first case in what would become
the largest Ebola outbreak to date. He lived in a small village in Guinea,
not far from the border of Sierra Leone and Liberia. In late December
2013, he became sick with fever, vomiting, and signs of hemorrhage.
The boy died after several days, followed closely by several otherfamily members. This strain of the virus is believed to have jumped
to humans from an infected fruit bat, its natural wildlife reservoir.1
Since the outbreak began nearly 18 months ago, the World HealthOrganization has documented 26,312 Ebola cases with 10,899
fatalities, as of April 29, 2015.2
The work to halt virus transmission has required input from a wide
range of experts, including clinicians, public health engineers, andepidemiologists. Yet, stopping the Ebola virus in its tracks will require
an understanding of the virus at its most basic level. Researchers are
now using next-generation sequencing (NGS) platforms like the HiSeq
and MiSeq Systems to understand the genetic structure of the virus.
Deep sequencing of samples from people infected with the virus hasrevealed some of its many secrets, including a rapid mutation rate.
Pardis Sabeti, PhD, a computational geneticist at Harvard University,and Christian Happi, PhD, a visiting professor of infectious disease atHarvard School of Public Health and Director of the African Center of

Excellence for Genomics of infectious Diseases (ACEGID), Redeemer’sUniversity, Ede, Osun State, Nigeria, have spent much of the last year
using the HiSeq System to understand how this virus spreads andchanges over time.

In the Center of the Storm
The pair first met at a conference in Senegal on computational
genomics, where they began looking for signals in the human genomethat might confer malaria resistance. Their work, however, revealed
a signal that Lassa fever, another hemorrhagic virus endemic toWest Africa, was placing a strong evolutionary pressure on humans.
Within a month, they put together an Institutional Review Board (IRB)
application and had a field site set up in Nigeria. They were deep into
this work when they first heard of the Ebola outbreak in Sierra Leone
in early 2014.
“We were in the middle of 1 of our weekly phone calls to discuss
ongoing Lassa work when we received the news,” Dr. Sabeti said.
“We immediately moved to conduct surveillance at our sites in Nigeria
and Sierra Leone, which were the best sites in this area to deal with ahigh-security virus. We knew that if the virus came to either of thesecountries that we would likely be dealing with it and we wanted to beprepared. When the first cases came in from Sierra Leone, we wantedto sequence the virus and understand if this was the same outbreakthat had been declared in Guinea.”

Team members from Nigeria, Senegal, and Sierra Leone who
established virology laboratories with the MiSeq Systemto monitor and track the presence of Ebola and otherpathogenic agents.
Researchers Christian Happi, PhD, and Pardis Sabeti, PhD, in alarge, open air church in Nigeria.

For Research Use Only. Not for use in diagnostic procedures.

May 2015
Their 7 years of work together on Lassa fever had placed them in aunique position to help. “These 2 diseases share several commonfeatures,” Dr. Happi said. “When the Ebola outbreak began, wewere prepared.”
Like Lassa, Ebola is a single-stranded RNA virus. Working with thevirus requires a maximum containment laboratory, also known as aBSL-4 lab. Ebola’s genetic material is RNA and therefore accumulates
mutations very quickly, because there’s no way for the viral machinery
to proofread sequences after replication. This fast mutation rate lets
the virus evolve rapidly and adapt to new situations. It also enables
researchers to track the virus over time by using the mutations asa guide.
When Ebola first struck, their lab was equipped with PCR technology,
and other equipment that they used for their research. Yet, the scaleof the outbreak and the work they wanted to do required genetic
sequencing—lots of it. Drs. Sabeti and Happi and their colleagues
evaluated several systems and ultimately chose the HiSeq 2500
System and the Nextera® XT Library Prep Kit.
“The HiSeq 2500 was by far the best performing sequencing system,
based on its depth of coverage and data quality,” Dr. Sabeti said. “We
tried about 5 or 6 different ways of preparing libraries and sequencing
the virus. We found that the Nextera XT Kit worked exceptionally welland gave us the best reproducibility between technical replicates.”

Tracing the Origins of the Ebola Outbreak
Dr. Sabeti’s team worked around the clock to sequence all thesamples and their hard work paid off. By late August, the team had
traced the origin and transmission of the 2014 West African Ebola
virus outbreak in unprecedented detail.3 They sequenced 99 Ebola
virus genomes from 78 patients at approximately 2000× coverage.
Their results revealed that Ebola first moved to West Africa from its
historic home in Central and East Africa in 2004. There was substantial
genetic variation as the virus moved from human to human, and evenas it multiplied within the same host. “Rather than 1 consensus viralsequence, we found thousands of different genetic snapshots of thevirus as it mutated in an individual,” Dr. Sabeti said.
The study also revealed that the virus likely only jumped to humans
from its animal reservoir 1 time, but that 1 spillover event created asustained chain of human-to-human transmission. In October 2014, a
separate group of scientists traced the Ebola transmission chain backto the toddler in Guinea.4“If we had seen that each of these outbreaks was independent,
and that they weren’t genetically related and coming from the same
evolutionary tree, then we might have thought these were different
infections from a natural reservoir and the result of different entries
from the environment,” Dr. Sabeti said. “However, these outbreaks
were closely related in time and the contact tracing supported
that. It suggested that this was a single event in a one long viraltransmission chain.”
These results confirmed the importance of contact tracing to preventthe spread of disease. Because the virus wasn’t being continually
reintroduced from the environment, epidemiologists knew that if they
could stop people from passing the virus to each other, then they
could stop the outbreak.

For Research Use Only. Not for use in diagnostic procedures.

Thousand Days – One Truth
The insights into Ebola were not gained without significant
cost to the physicians and scientists on the ground during
the outbreak. More than 800 health care workers were
infected with the virus and 492 have died, including someof the people Drs. Sabeti and Happi worked with. Out ofthat pain came a song that means a lot to Dr. Sabeti andher research team in West Africa.
Her team included a group of 11 scientists from Nigeria andSenegal, who traveled to Sabeti’s lab during the outbreakfor training in genomics. Sabeti, a gifted musician andvocalist, enjoyed singing with them.
“The women all have beautiful voices,” Dr. Sabeti said.
“When they came to visit, we’d gather and sing once a week.
Even though we were working hard and I often wasn’tsleeping, I promised to keep this date. It was in the midst of
1 of our weekly sessions that the inspiration came. I lookedat the faces of these women and the song just wrote itself.”

Watch the video.

The deep sequencing data provided Drs. Sabeti and Happi with otherinsights. “With NGS, you get high-resolution data and can see morethan just the common variants,” Sabeti said. “If we’d had only thecommon variants, we would have missed a lot and it would have been
hard to see who was infecting whom at a high scale. At 1% frequency
or lower, you can see all the rare things that are percolating and canbegin to understand the transmissions. NGS provided us with thedepth of information we needed to understand the human-to-humanviral transmission chain.”Open Data Allows Deeper Exploration
The reams of sequencing data that Drs. Sabeti and Happi released
allowed many other groups to advance our understanding of how
the outbreak evolved and informed the development of therapeutics
to combat outbreaks in the future. In January 2015, they worked
with a team at the U.S. Army Medical Research Institute of Infectious
Diseases (USAMRIID) investigating how Ebola’s rapid mutation rate
could potentially impact the efficacy of currently available treatments.5
These include sequence-based therapeutics such as small interfering
RNAs (siRNAs), phosphorodiamidate morpholino oligomers (PMOs),and antibodies that target genes and proteins within the Ebola virus.
For each outbreak, the specific targets of these drugs will need to bemodified to make sure that candidate treatments and vaccines arestrain-specific.
Sequencing with the HiSeq System also enabled examination of thepatient samples for other microbes, and thus new discoveries about
factors that might help people survive an Ebola infection. Led by theUniversity of Wisconsin, the team found that individuals carrying thepegivirus GB virus-C, which is known for its ability to modulate theMay 2015
immune system, were significantly less likely to die of Ebola than
individuals who weren’t coinfected with GBV-C.6 “These are amazing
insights you can only find with NGS,” Dr. Sabeti said.

New Laboratories to Monitor Viral Outbreaks
With financial support from USAID and Illumina, Drs. Sabeti andHappi obtained a MiSeq System in January 2015 as the foundation of
Nigeria’s first next-generation virology lab. They also obtained a MiSeq
system for a laboratory in Senegal, and collaborated with USAMRIID
on their work to establish a laboratory with a MiSeq System in Liberia.
Illumina will soon provide a MiSeq System for a virology laboratory in
Sierra Leone. For them, the MiSeq System is an obvious choice.
“Of all the NGS systems, the MiSeq System is a standout,” Dr. Sabetisaid. “There is no other NGS system right now that has this kind ofcapacity to move into the field. It has very high efficiency and you
get great reads and rapid turnaround. In some places, we have toconsider power efficiency. The MiSeq is a just a beautiful system thatworks very well.”“Based on our own interaction, the MiSeq System is also very userfriendly,” Dr. Happi added.
They are also continuing to use the Nextera XT Library Prep Kits. “Weuse the Nextera XT kit because it has good efficiency and very goodaccuracy,” Dr. Sabeti said. “We have made a few modifications to thekit, enabling us to remove more of the human material so that we canenrich as much of the viral material as possible.”
They hope to use the MiSeq System to gain a better understandingof the whole range of viruses and pathogens that affect people living
in West Africa. Currently, the lab is focusing on Ebola sequencing,although they plan to perform sequencing on Lassa fever
samples shortly.
“Pardis and I are always moving forward with technology, making surethat we’re using the latest core technology to understand disease
and health problems in Africa,” Dr. Happi said. “By bringing together
knowledge, know-how, and technology, we’re giving people the ability
to start looking inward to solve problems in this region. Now that wehave the MiSeq System, I think it’s important that we enable African
scientists to start using this technology and start answering questions
that are important to people in Africa.”“The Nigeria laboratory is a landmark facility,” Dr. Sabeti said. “Itis developing into a training center, with a graduate program that
Christian is in charge of for West African students. There’s a strong
interest forming in West Africa and beyond in understanding thegenomics in infectious disease. Christian is leading that effort here.”

A Constantly Mutating Foe
The Ebola virus strain responsible for the 2014 outbreak is still
mutating. In addition to the 99 strains sequenced early during theoutbreak, the team has since cataloged another 150 Ebola genomes
and made them available in a public database.7
Even as the outbreak eventually winds down, it won’t necessarily be
the end for Ebola sequencing. Dr. Sabeti says that scientists need tokeep on top of what viral strains are in play and monitor any changes
that are occurring. Both researchers believe that NGS will open a newchapter in the never-ending fight against viruses.
For Research Use Only. Not for use in diagnostic procedures.

References
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6.
7.

Mari Saéz A, Weiss S, Nowak K, et al. Investigating the zoonotic origin of
the West African Ebola epidemic. EMBO Mol Med. 2014; 7(1): 17-23.
WHO Ebola Situation Report, April 29, 2015: http://apps.who.int/ebola/current-situation/ebola-situation-report-29-april-2015.
Gire SK, Goba A, Andersen KG, et al. Genomic surveillance elucidates
Ebola virus origin and transmission during the 2014 outbreak. Science.
2014; 345 (6202): 1369–1372.
Baize S, Pannetier D, Oestereich L, et al. Emergence of Zaire Ebola Virus
Disease in Guinea. N Engl J Med. 2014; 371(15): 1418–1425.
Kugelman JR, Sanchez-Lockhart M, Andersen KG, et al. Evaluation of thePotential Impact of Ebola Virus Genomic Drift on the Efficacy of SequenceBased Candidate Therapeutics. MBio. 2015; 6(1): pii: e02227-14.
Lauck M, Bailey AL, Andersen KG, et al. GB Virus C coinfections in westAfrican Ebola Patients. J Virol. 2015; 89(4): 2425–2429.
NCBI Ebolavirus database: http://www.ncbi.nlm.nih.gov/genomes/VirusVariation/Database/nph-select2.cgi?cmd=database&taxid=186536

Learn more about the Illumina systems and products
mentioned in this article, and our partnership with theBroad Institute of MIT and Harvard, and USAID to combat
the Ebola epidemic in West Africa:••••

HiSeq System, www.illumina.com/systems/hiseq_2500_1500.html
MiSeq System, www.illumina.com/systems/miseq.html
Nextera XT DNA Library Prep Kit, www.illumina.com/products/
nextera_xt_dna_library_prep_kit.html
USAID, Broad Institute, and Illumina Form a Public-Private
Partnership Combating the Ebola Epidemic in West Africa,investor.illumina.com/phoenix.zhtml?c=121127&p=irolnewsArticle&ID=1990982

May 2015

Illumina • 1.800.809.4566 toll-free (US) • +1.858.202.4566 tel • techsupport@illumina.com • www.illumina.com
For Research Use Only. Not for use in diagnostic procedures.
© 2015 Illumina, Inc. All rights reserved. Illumina, MiSeq, Nextera, and the pumpkin orange color are trademarks of Illumina, Inc. and/or itsaffiliate(s) in the U.S. and/or other countries. Pub. No. 1270-2015-001 Current as of 14 May 2015