July 2015

From Arrays to NGS: How Chromosomal Genetics
Evolved From Structure to Disease
Researchers from the MGZ Medical Genetics Center are using the Infinium® CytoSNP-850K
BeadChip and the TruSight® Cancer Panel to analyze chromosomal abnormalities.
IntroductionMore than 100 years after chromosomes were first made visible under
the light microscope, scientists made the discovery that humans
possessed 46 chromosomes, rather than the 48 previously assumed.1
Making up the programming core of the human body, the DNA
these chromosomes contain is responsible for what makes each ofus unique. However, the slightest chromosomal change can lead toharmful consequences. An extra chromosome can trigger congenital
disorders such as Down syndrome. An unbalanced exchange of genetic
material between chromosomes (eg translocation) can result in severe
genetic disorders.
The myriad of cytogenetic alterations and their impact on humandisease was intriguing to Udo Koehler, PhD, now Laboratory Director
at MGZ Medical Genetics Center. Originally trained in primate evolution,
he was fascinated by how small cytogenetic changes could have suchprofound consequences. It motivated him to shift his focus to humangenetics studies and the development of cytogenetic diagnostics. Anna
Benet-Pagès, PhD, also a Laboratory Director at MGZ found herself ina similar situation. Originally trained in biochemical genetics, she wasdrawn to the possibility of applying her expertise to human health.
Drs. Koehler and Benet-Pagès are part of a growing team performing
cytogenetic analysis, next-generation sequencing (NGS), and researchstudies on human genetic disorders at MGZ. Founded in April 2000, MGZ
has expanded from 1 human geneticist and 15 technicians to a team of
11 geneticists and more than 90 staff members, 20 of whom have PhD
degrees. Over the course of their scientific careers, Drs. Koehler andBenet-Pagès have witnessed numerous technological advances, fromearly banding techniques to novel microarrays, and NGS.

Leveraging Novel Technologies to Improve Resolution
Dr. Koehler began his studies in the prePCR and presequencing eraof genetics. “We used conventional karyotyping by G-banding andfluorescence in situ hybridization (FISH) to study chromosomes,”
Dr. Koehler said. Conventional karyotyping is the process of staining
chromosomes with Giemsa stain (hence the name, G-banding) for
visualization under light microscopy. FISH uses fluorescently labeled
DNA probes to detect and localize the presence or absence ofspecific DNA sequences on chromosomes.
“We performed every FISH technique imaginable,” Dr Koehler stated.
“Single-locus FISH, multiplex FISH, subtelomeric FISH, microdeletion
FISH, and interphase FISH. We sampled blood, buccal smear, andurine to test for mosaics. It was a lot of microscope work.”
The introduction of the chromosomal microarrays in the early 2000s
represented a paradigm shift in cytogenomics studies. Chromosomal
arrays are used to scan the entire genome to identify small copy
number variations (CNVs). At the start of the microarray era, arrays
had a resolution of 1 megabase (1000 kilobases), which has sinceincreased 100-fold or more. At a resolution of 10 kilobases or higher,scientists can feasibly detect copy number variants and alterations at
the single gene, or even single exon level.

Lab Directors Udo Koehler, PhD, and Anna Benet-Pagès, PhD, are part of an interdisciplinary team performing cytogenetic analysis,next-generation sequencing, and research studies at MGZ Medical Genetics Center in Munich, Germany.

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

July 2015
Using chromosomal arrays, researchers began discovering copy
number variations rapidly, leading to the identification of newsyndromes. “It was fascinating to take part in the evolution ofmicroarray analysis,” Dr. Koehler said. “Arrays enabled me toidentify the smallest genomic copy number variant, which wouldn’t
have been detected by conventional karyotyping. It’s now a firsttier approach for identifying developmental delay in children.” Thestraightforward and clear results from chromosomal microarray
analysis “make the lab staff happy,” Dr. Koehler added. “They
don’t have to look through a microscope anymore, which istime consuming and requires years of experience.”

The Right Tool for the Job
Continuous growth has enabled MGZ to acquire technologies that
support new cytogenomic approaches. It started using microarrays
and later added an iScan® System. MGZ became the first German
laboratory to use the Infinium CytoSNP-850K BeadChip after it wasintroduced in 2013. They use BlueFuse® Multi Software to analyze
the data.
“The CytoSNP-850K BeadChip offers accurate cytogenomic testing,”
Dr. Koehler said “I like that it is SNP-based, which means there arefewer false-positive copy number variants. With no labeling artifacts,
the interpretation of results is much clearer, mosaics can be detected
better, and I can clearly see the number of copies in the B allele chart.”“When I look at the data using the BlueFuse Multi Software, there’s
rarely any variant I can’t match with the databases,” Dr. Koehler added.
“I like the links to the genome browsers and the zoom in and query
capability to compare regions with other results. Creating batch files iseasy, too. I can import the lab’s entire database to the BlueFuse Multi
Software with just one click. I can analyze more patients in a shorter
timeframe, which is very helpful for the lab workflow.”
About 90% of the samples MGZ analyzes with the CytoSNP850K BeadChip are postnatal. These analyses help identify thecauses of autism spectrum disorders, intellectual disability (ID),dysmorphic features, or neurological disorders such as epilepsy.
MGZ also performs prenatal analysis with conventional karyotyping,chromosomal microarrays, and noninvasive prenatal testing (NIPT)
for chromosomal aneuploidy (abnormal number of chromosomes).

Assessing the Genetics of Cancer
The CytoSNP-850K BeadChip and traditional cytogenetic
techniques aren’t the only genetic analysis tools used by MGZ. In
2012, Dr. Benet-Pagès brought NGS to the laboratory. She began
using Illumina sequencing systems in 2007 during her postdoc,
beginning with the Genome Analyzer and later moving to theHiSeq® System. At MGZ, she uses the MiSeq and NextSeq®
500 Systems to run a large number of custom panels to identify
variants associated with rare hereditary diseases and the TruSight
Cancer Sequencing Panel, which targets 94 genes with suspectedassociations to common and rare cancers. The sequencing panel

also includes 284 SNPs suspected to correlate with cancer throughgenome-wide association studies (GWAS).
“The TruSight Cancer Sequencing Panel is a test method that anyone
can adapt easily in their laboratory,” Dr. Benet-Pagès said. “It’s easy toimplement in automated workflows and performs with high repeatability
and reproducibility. The TruSight Cancer Panel enables us to analyze moregenes in a single workflow than previous technologies. The advantage isthat we can identify potential mutations in cancer associated genes notprimarily tested.”“The TruSight Cancer Sequencing Panel delivers results in 6–7 days,”
Dr. Benet-Pagès added. “That includes the whole workflow process,from sample acceptance, DNA extraction, sample preparation,
sequencing, and data interpretation, through to data reporting.”

The Future of Cytogenetic Analysis
Drs. Koehler and Benet-Pagès would like their cytogenetics andNGS labs to work together more because the techniques arecomplementary. “Array-based technologies are useful in detecting
CNVs such as large deletions and duplications, but not in detecting
single nucleotide variants (SNV),” said Dr. Koehler. “NGS enables thedetection of unexpected aberrations, such as SNVs, which aren’t wellcharacterized. Pairing these 2 methods—dose analysis for CNVs andsequence analysis for SNVs—will determine the genomic basis for anincreasing number of inherited disorders. This approach would benefit
from a single software solution, perhaps BlueFuse Multi Software.”

Reference
1.

Genetic Timeline, National Genome Research Institute web site.
www.genome.gov/Pages/Education/GeneticTimeline.pdf.
Accessed May 12, 2015.

Learn more about the Illumina products andsystems mentioned in this article:
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Infinium CytoSNP-850K BeadChip, www.illumina.com/products/
infinium-cytosnp-850k.html
TruSight Cancer Panel, www.illumina.com/products/trusight_cancer.htmlBlueFuse Multi Software, www.illumina.com/clinical/clinical_informatics/bluefuse.html
iScan System, www.illumina.com/systems/iscan.html
HiSeq System, www.illumina.com/systems/hiseq_2500_1500.html
MiSeq System, www.illumina.com/systems/miseq.html
NextSeq System, www.illumina.com/systems/nextseq-sequencer.
html

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, BlueFuse, HiSeq, iScan, MiSeq, NextSeq, TruSight, and the pumpkin orange color are
trademarks of Illumina, Inc. and/or its affiliate(s) in the U.S. and/or other countries. Pub. No. 1570-2015-008 Current as of 02 October 2015