October 2015

Unmasking the Viral Etiology of Cancer andImmune Disease
Karolinska Institutet researchers use Illumina sequencers to identify novel HPV types associatedwith non-melanoma skin cancers.
Introduction
Despite decades of research, the causes of cancer seem to many tobe as mysterious and unresolved as ever. Yet, there are certain typesof cancer where the drivers of malignancy have emerged into stark
focus. Scientists know that human papillomavirus (HPV) causes 99%of cervical cancer cases1. HPV is a large DNA virus that establishes
infections in keratinocytes of the mucous membranes, as well as
the skin where it causes squamous cell papilloma or warts. If HPV isresponsible for most cervical cancer cases, could it be responsible forcertain skin cancers?
The strong link between HPV and cervical cancer prompted Emilie
Hultin, PhD, to wonder if there was a connection between HPV andnonmelanoma skin cancer. A geneticist at the Karolinska Institutet in
Sweden, Dr. Hultin’s HPV research has spanned the transition frommicroarrays to next-generation sequencing (NGS), revealing novel
HPV types present in nonmelanoma skin cancer. Relying on Illumina
HiSeq®, MiSeq®, and NextSeq® Systems, her research underscores
the ability of NGS to unravel some of medicine’s biggest mysteries.

Unlocking HPV Secrets
Dr. Hultin began her science career studying chemical engineering at
the Royal Institute of Technology (KTH) in Stockholm. During her PhD
studies at KTH, she developed new technologies and methods forarray-based SNP genotyping and HPV typing. After a 2-year sojournworking in industry, Dr. Hultin moved to the Karolinska Institutet in2011. She is currently a research coordinator in the lab of Joakim
Dillner, MD, PhD, where she runs the group’s deep sequencing lab.

Dr. Hultin stated. “NGS enabled us to sequence all the DNA in skinsamples, making it possible to detect novel HPV types.”
Dr. Hultin’s arrival in Dr. Dillner’s lab coincided with their first purchaseof an NGS sequencer, the Roche 454 System. The team later useda core facility’s Thermo Fisher Scientific Ion Torrent sequencing
system. The team soon realized that neither system provided thesequencing depth to find novel viruses. “The viral signals werebeing swamped out by the overwhelming amount of skin cell DNA
in the samples,” Dr. Hultin said. “Only about 0.2% to 0.5% of all thesequencing reads were actually viral DNA—the rest was human. We
also wanted a sequencing system that could multiplex, enabling us
to sequence cost-effectively. It’s too expensive to run one sample persequencing run.”

MiSeq System Uncovers HPV Diversity in Skin
The MiSeq System offered the perfect alternative, meeting their deep
sequencing requirements, and capable of multiplexing 96 samples
per run. They conducted a head-to-head comparison of the MiSeq
platform, 454, and Ion Torrent systems to see how each system
performed. The MiSeq System delivered, providing more reads anda 1,000 fold more sequencing depth than the other systems. Yet,the study was much more than a sequencer showdown. Dr. Hultin
and her team used the systems to conduct a set of experiments
looking for novel viruses in 142 different skin samples from
immunosuppressed individuals with actinic keratosis, basal, andsquamous cell carcinomas.2Drs. Hultin and Dillner are interested in identifying viruses associatedwith different human diseases, especially cancer. Their work has
focused mainly on detecting causal links between HPV and cervicalcancer. In the genital tract, certain types of high-risk HPV viruses aremuch more likely to turn cervical cells cancerous, whereas low-risk
HPV viruses are limited to causing genital warts. Because HPV canalso infect skin, they decided to investigate whether the HPV-cancer
link might also include nonmelanoma skin cancers. They decided toexclude melanoma from their studies because of its strong association
with sun exposure.
“We knew that there were many HPV strains on the skin, but weren’t
sure whether they could cause nonmelanoma cancers,” Dr. Hultin said.
“Establishing the presence of a link required identifying small amountsof HPV DNA in skin samples. That was not an easy task.”
Because they didn’t know what they were looking for, they used NGS
for their studies. “Microarray technology is directed toward known
DNA sequences, so we couldn’t use it to identify novel viruses,”

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

Dr. Emilie Hultin is a Research Coordinator at the Karolinska
Institutet in Sweden.

October 2015
Study samples consisted of whole genome amplified and PCRamplified sample pools. “There’s an advantage in sequencing
unbiased and biased sample pools,” Dr. Hultin said. “When wesequence unbiased whole genome amplified material we are able todetect more HPV types, but we also get a lot of human reads. PCR
amplified samples enable us to obtain more HPV-only reads. However,
we’ll miss some HPV types that are not complimentary to the primers
we are using.”
The 2 sample pools required the use of 2 different Illumina library
prep kits. “We used the Nextera® DNA Library Preparation Kit for
the 82-sample whole genome amplified pool and were able toprepare libraries from all these samples in just 1-2 days,” Dr. Hultin
said. “It was convenient to analyze a full plate at the same time as weprepared the samples.”“The MiSeq System identified HPV
types that were not found using
the 454 and Ion Torrent systems.”“We also had samples that were amplified with specific PCR primers,
creating amplicons of about 450 bp,” Dr. Hultin added. “That was theperfect size, so we used the TruSeq® Nano DNA Library Preparation
Kit for these samples. We eliminated the fragmentation, end-repair,and size selection steps so we could ligate the sequencing adaptersto the ends of the PCR products immediately. The Illumina library
kit protocols and MiSeq workflow reduced our hands-on time in thelab dramatically.”
The study found an unexpected level of diversity, with no less than 396
HPV types, containing 229 putative virus types that had never before
been identified. In contrast, a sequencing study performed earlier withthe 454 and Ion Torrent systems identified 273 different HPV types,
and only 47 unknown viruses.

Using the HiSeq and MiSeq Systems, Dr. Hultin led a follow-up study
that looked for HPV in 91 skin samples that were a combination of FFbiopsies and FFPE samples from keratoacanthomas, actinic keratosis,
and basal cell carcinomas.3 Multiplex, paired-end sequencing onthe HiSeq System using dual indexes enabled them to sequence
the samples using a non-biased approach. They found 4 previously
unknown putative HPV types, including HPV197, which was highly
present in the different cancer samples. HPV197 is only 75 percent
identical to the most closely related type of HPV. Its primer regions
possessed many nucleotide differences that would have prohibited
PCR amplification.
“HPV197 would never have been discovered without using an
unbiased method,” Dr. Hultin said. “This virus is abundant among
these nonmelanoma skin cancer samples, so it seems likely that it is avery important skin HPV type.”

A Shift to the NextSeq 500 System
In June 2014, Dr. Hultin added a NextSeq 500 System to the lab andhas been thrilled with its performance. Regardless of whether they’re
doing PCR amplification or not, Dr. Hultin’s team has found that
the NextSeq 500 System is perfect for their studies. “The NextSeq
500 System enables us to sequence FFPE samples effectively, andassemble novel viruses from FF biopsy samples,” Dr. Hultin said. “Itoffers greater sequencing depth and an ability to sequence pools of
96 samples. We’ve shifted primarily to the NextSeq 500 System forour studies, using the MiSeq System for confirmation or pilot studies.”
The team’s bioinformatics pipeline is optimized for paired-end
sequencing data. Given that viruses like HPV can integrate themselves
into the host genome, paired-end sequencing with the NextSeq 500
System enables Dr. Hultin to identify locations where the virus has
integrated. “We can look at the pairs and see if one pair is alignedto the virus and the other pair is aligned to the human genome,”
Dr. Hultin said.

“We thought that we were going to find a few novel HPV types,
because there hadn’t been many studies of skin HPV,” Dr. Hultin said.
“We didn’t expect to find such extreme diversity of cutaneous HPV
types. The MiSeq System identified HPV types that were not foundusing the 454 and Ion Torrent systems.”“HPV197 would never have been
discovered without using anunbiased method.”

After 2 years using the 454 and Ion Torrent systems, Dr. Hultin madethe switch to the MiSeq System, enabling her to make greater strides
in her research. “It wasn’t until we switched to the MiSeq Systemthat we had the sequencing depth that we needed, yielding data thatenabled us to identify novel HPV types, Dr. Hultin said. “The resultsof that study marked a fundamental change in our understanding ofHPV diversity.”Dr. Hultin is thrilled with the performance of the NextSeq 500 Systemand how easy it is to use. “The MiSeq System training was easy andwe couldn’t believe that sequencing could get any easier,” Dr. Hultin
said. “Then we were trained on the NextSeq 500 System and realizedthat Illumina had indeed made sequencing a bit easier. We almost
didn’t need any training at all.”

Short-Read Sequencing Identifies Novel HPV TypeThe team continued their research, sequencing serum, fresh frozen(FF) biopsies, and formalin-fixed paraffin embedded (FFPE) skinsamples. Because the DNA in FFPE samples is highly degraded,
they thought that longer reads would be better in identifying novel
HPV types. But as they worked, they found that shorter pairedend sequencing (151 bp) actually gave them the best results forFFPE samples.

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

Viral Etiology in Cancer and Immune System Diseases
NGS has opened up a window into the number of HPV viral types
that exist. “A couple of years ago, there were about 100 different
HPV types known,” Dr. Hultin stated. “Now more than 200 types havebeen identified.”
Their success with HPV has enabled the researchers to look more
broadly at the role of viruses both in cancers, and in diseases
and disorders that impact the immune system negatively. Nobel

October 2015laureate Harald zur Hausen, MD, who first discovered the role ofHPV, has proposed that immunosuppressed individuals are more
likely to get cancers that are caused by viruses, because their own
immune systems are no longer able to keep the infections in check.
An example is AIDS, where patients are at much higher risk ofnonmelanoma skin cancers such as Kaposi’s sarcoma.

“The NextSeq 500 Systemenables us to sequence
FFPE samples effectively, andassemble novel viruses from FF
biopsy samples.”Dr. Hultin and her team have been looking for viral signatures incancers associated with immunosuppression in the hopes of finding
a potential viral link. They are also conducting a study on multiple
sclerosis (MS) to see if there’s evidence of a viral etiology. Dr. Hultin
has obtained serum samples from MS patients taken several years
before their diagnosis to identify viruses that could be linked to MS.
The first NextSeq 500 System sequencing runs have been completed
and the group is working on the confirmation phase.
Although the role of HPV in cervical cancer has become clear,scientists are just beginning to understand the role of HPV and otherviruses in the development of cancer. Dr. Hultin believes that many
more types of cancer might have a viral etiology, even if viral infection
doesn’t automatically cause cancer down the line. With the help ofNGS, she believes she will ultimately find these signatures.

References1.

Walboomers JM, Jacobs MV, Manos MM, et al. Human papillomavirus
is a necessary cause of invasive cervical cancer worldwide. J Pathol.
1999;189:12–19.
2. Bzhalava D, Mühr LS, Lagheden C, et al. Deep sequencing extends thediversity of human papillomaviruses in human skin. Sci Rep. 2014; 4: 5807.
3. Arroyo Mühr LS, Hultin E, Bzhalava, et al. Human papillomavirus type 197 iscommonly present in skin tumors. Int J Cancer, 2015; 136(11): 2546–2555.

Learn more about the Illumina products and systems
mentioned in this article:•MiSeq System, www.illumina.com/systems/miseq.html•HiSeq System, www.illumina.com/systems/hiseq_2500_1500.html•NextSeq System, www.illumina.com/systems/nextseq-sequencer.html•Nextera DNA Library Preparation Kit, www.illumina.com/products/nextera_dna_library_prep_kit.html•TruSeq Nano DNA Library Preparation Kit, www.illumina.com/products/truseq-nano-dna-library-prep-kit.html

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

October 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, HiSeq, MiSeq, Nextera, NextSeq, TruSeq, and the pumpkin orange color are trademarks ofIllumina, Inc. and/or its affiliate(s) in the U.S. and/or other countries. Pub. No. 770-2015-023 Current as of 15 October 2015