October 2014

Sequencing Challenging FFPE Cancer Samples toIdentify Cancer Driver Genes and Novel Biomarkers
Yasser Riazalhosseini, Ph.D., leverages key features in the Nextera® Rapid Capture Exome library
prep kit to preserve integrity of low-input DNA from low-quality samples.
Introduction
Biopsies or tissue samples from cancer patients are routinely
stored in formalin-fixed, paraffin-embedded (FFPE) tissue
blocks. Sequencing these samples has proved challenging,
because the process partially degrades and limits the amountof genomic material. Advanced approaches, such as nextgeneration sequencing (NGS), generate high-quality genomic
data and are suitable for analyzing these valuable samples.
Yet, some sequencing preparation methods further degrade
the already low-quality FFPE sample DNA.
Sequencing challenging samples is part of the job for
Yasser Riazalhosseini, Ph.D., an Assistant Professor in theDepartment of Human Genetics at McGill University andprincipal investigator at McGill University and Genome Quebec
Innovation. He studies unique forms of cancer to translatefindings from basic science into clinical application.
Until recently, he had a backlog of stored samples yet to
be analyzed due to the difficulty of working with the FFPE
format and limited amount of DNA. Dr. Riazalhosseini is now
accessing these samples by leveraging key features in theNextera® Rapid Capture Exome (NRCE) library prep kit toperform exome sequencing on a HiSeq® system. Working
with at least 50 ng DNA, Dr. Riazalhosseini can preserve
DNA integrity and analyze disease-causing mutations andprognostic signatures.
iCommunity spoke with Dr. Riazalhosseini to learn more about
his research and the benefits of using the NRCE library prep kitfor exome sequencing of low-quality samples.
Q: What are the goals of your research?
Yasser Riazalhosseini (YR): My research involves generating
and analyzing large cancer genomic data sets to understandmolecular mechanisms involved in carcinogenesis andprogression, and to identify molecules with potential
clinical applications. In my lab, we rely on the generation
and analysis of high-throughput genomic sequencing andtranscriptome data.
Q: What features of the NRCE kit have had the greatest
impact on your research?
YR: We’ve been very successful using the NRCE Kit. Its main
advantage is that it can accommodate small DNA samples.
It opens the door for us to study samples, especially biopsy

Yasser Riazalhosseini, Ph.D., is an Assistant Professor in the Department of
Human Genetics at McGill University, and principal investigator at the McGill
University and Genome Quebec Innovation Center in Montreal.

samples, that are well-characterized clinically, but that have
limited DNA. With the NRCE Kit, almost all tumor types—
including ones stored as fresh-frozen or in FFPE format—canpotentially be characterized. I’m using the Nextera kit for all myprojects and in my collaborations at the moment, two of whichhave already come to publication.
Q: What was the first study that you published using theNRCE kit?
YR: The first study, involving characterization of small cell
carcinoma of the ovary hypercalcemic type (SCCOHT), wasled by Dr. William Foulkes at McGill University and published inNature Genetics earlier this year.1 SCCOHT are undifferentiatedtumors in the ovary, usually occurring in women before the ageof 40. We obtained tumor samples from colleagues in Europe,
Australia, and Canada who never had a chance to look at thegenomes of these samples. They provided FFPE samples, notfresh-frozen samples, which makes it difficult to analyze withNGS. We used Nextera technology to generate the exome
libraries from DNA isolated from these FFPE samples andsequenced them on a HiSeq system.
Q: What were the findings from the ovarian cancer study?
YR: We identified deleterious mutations in a gene called
SMARCA4. We were very surprised to see that almost all
patients had a deficiency in SMARCA4. It was validated
by analyzing the gene product at the protein level in tissue
samples from the same patients. For the first time, we could
characterize these types of tumors and begin to understandthe biology behind this cancer.

October 2014
Q: What type of cancer was the focus of your second
study?
YR: It was a breast cancer study led by Dr. Michael Hallett
at McGill University. We collaborated on this project and apaper reporting results of this study has been published in thejournal Cell Reports.2 The aim of the study was to validate theprognostic signatures resulting from the analysis of thousandsof breast cancer samples. It’s known in the literature thatbreast cancer heterogeneity influences the prognostic
markers that are generalized to a patient. As a result, further
characterization is required before extending the validity of aprognostic signature to all patients.

“With the NRCE Kit, wegenerate libraries using low
DNA amounts that result inhigh-quality sequencing.”
Q: What were the findings of the breast cancer study?
YR: The team compared the prognostic signatures across alarge collection of patients. Since some patients were almost
always mispredicted, they hypothesized that such patients—
especially those that ultimately experienced a recurrence—might harbor a high degree of tumoral heterogeneity. Tovalidate this observation, we sequenced exomes of theseheterogeneous breast tumors. Using the NRCE Kit we wereable to sequence exomes on the HiSeq System and validate
the team’s hypothesis.
Q: How does the ability of the NRCE Kit to accommodate
low-input DNA enable your studies?
YR: The NCRE Kit accepts 50 ng of DNA to generate an
exome library for sequencing. This is critical, because itenables us to work with unique tumor samples, particularly
biopsies from patients with specific conditions. We could notanalyze the exomes of many samples before because of theminute amount of DNA that we had, and the absence of atechnology that could generate an NGS library from such low
DNA amounts. With the NRCE Kit, we generate libraries using
low DNA amounts that result in high-quality sequencing. We’ve
tried it on different samples and continue to be successful.
Q: How is the tagmentation step in the NRCE protocol
beneficial to your research?
YR: Instead of shearing the DNA, or chopping it into shorter
fragments to generate the library, the Nextera kit uses
transposase enzymes to fragment the library. This is criticalbecause when we use low quality FFPE samples, the DNA isalready quite degraded. The shearing just destroys the DNA
further, resulting in poor libraries. By leveraging the Nextera
tagmentation step, we can work with FFPE samples andgenerate high-quality libraries.

Q: What did you learn working with the transposase
enzymes?
YR: The enzymatic tagmentation is sensitive to the amountof input DNA. The size of the final library is determined by theratio of DNA to transposase enzymes. The enzyme can also
behave differently in different environments. I took advantageof this by changing the amount of input material to produce
libraries with different characteristics, such as the insert size,
which is important for sequencing.
Q: How do you measure assay performance?
YR: We check the quality of sequencing by looking at
the number of reads and the amount of data generated,
performing further analyses after alignment to measure thespecificity of capture for the targeted exome. We target anaverage 70× depth of coverage for whole-exome sequencing.
We can achieve this by sequencing three exomes on one
lane of the HiSeq System. The Nextera protocol is sensitive inhandling. We found that when we follow the library generation
steps carefully, we get homogeneous results for all samples.
Q: How long did it take you to implement the NRCE Kit into
your workflow?
YR: My lab collaborated with colleagues at the NGS unitof McGill University and the Genome Quebec Innovation
Center to evaluate the beta version of the NRCE Kit. Thelibrary preparation protocol is not complicated. Due to theinitial enzymatic step, it’s a sensitive protocol in comparison
to others.
Following the initial tests, McGill University’s NGS unit
established the NRCE pipeline quickly, and they are optimizing
the procedures on robots for high-throughput use of the kit.
My group examined different methods for DNA isolation fromFFPE samples to identify a protocol that induces minimum
damage to DNA during isolation, while not interfering with thelibrary generation steps. With that process in place, it takes amatter of days to generate the libraries.

“Using the NRCE kit, we areconducting similar studies onother less-studied cancers
to understand the underlying
genetic aberrations.”
Q: Did the NRCE Kit support your ability to generate cancer
study results that translate clinically?
YR: I think the best example is the SCCOHT study. In thiscancer study, SMARCA4 gene was recurrently mutated in all
the patients included in the study. It demonstrates that thistype of cancer has one main driver. SCCOHT is difficult todiagnose pathologically. The ability to identify the driver geneOctober 2014
in the patient genome has implications for diagnosis andclinical management in the future. Using the NRCE kit, we areconducting similar studies on other less-studied cancers tounderstand the underlying genetic aberrations.

References
1.

Witkowski L, Carrot-Zhang J, Albrecht S, et al. Germline and somatic
SMARCA4 mutations characterize small cell carcinoma of the ovary,
hypercalcemic type. Nature Genetics 2014; 46(5): 438-43.

2.

Tofigh A, Suderman M, Paquet E, et al. The prognostic ease and difficultyof invasive breast carcinoma. Cell Reports 2014. DOI: http://dx.doi.
org/10.1016/j.celrep.2014.08.073.

October 2014

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