Cytogenetics to Cytogenomics: An Introduction toGenomic Technologies

www.illumina.com/cytogenetics

Table of Contents
I. From Microscopes to Genomes

3

II. Genomic Technologies for Cytogenetics
Arrays4

Next-Generation Sequencing

5III. What is Next-Generation Sequencing?

6The Basic NGS Workflow

6

Multiplexing

8

IV. Summary

8

V. Glossary

9

VI. References

9

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

I. From Microscopes to Genomes
To understand the role that chromosomal variations play in both constitutional disorders and cancer, cytogeneticanalysis is an integral part of current genomic medicine. Chromosomal abnormalities, including aneuploidies,deletions, duplications, and rearrangements, may result in misregulation of gene expression or generation of novel
proteins. Such abnormalities are a common cause of cancer, infertility, and various congenital disorders such as
Down syndrome, DiGeorge syndrome, and cri du chat syndrome.1
Traditional cytogenetic studies use karyotyping and/or fluorescence in situ hybridization (FISH) to analyze
chromosomes. These approaches have been instrumental in identifying major chromosomal abnormalities such asmonosomies, trisomies, chromosomal rearrangements, and large deletions or duplications. However, these methods
are limited by low resolution or narrow target range. For example, karyotyping is capable of detecting only large
chromosomal changes (typically > 5 Mb).2 It is a subjective technique, and detection of abnormalities often depends
on the expertise of the analyst. FISH and real-time quantitative PCR (qPCR) are highly targeted approaches, but
require prior knowledge of the abnormality.
Due to recent advancements in high-throughput technologies, cytogeneticists are using genomics tools to
overcome these limitations when studying chromosomal abnormalities. Compared to karyotyping, arrays offer
a more reliable method for identifying copy number variations and unbalanced chromosomal rearrangements.
Because it offers wider genome coverage with higher resolution, array-based research is often used as a firstline of testing to identify both small and large alterations associated with congenital disorders and various
cancer subtypes.3–6
The most recent evolution of genomic technology is next-generation sequencing (NGS), a high-throughput process
for determining DNA sequences. Sequencing can be used to explore variants detected by arrays or to identify
small variants missed by other methods. By providing a base-by-base view of the genome, NGS can identify single
nucleotide variants (SNV), small structural changes, and balanced translocations, increasing information while
decreasing costs with a genome-wide view of variation.

“Now that we have whole-genome tools such as microarrays and NGS, we can analyze the entire
genome from a few malignant cells at a resolution that allows us to identify abnormalities and lookfor rearrangements with a very high degree of precision.”—Trilochan Sahoo, MD, Director of Cytogenetics, CombiMatrix7

Cytogeneticists can use complementary array and NGS technologies for thorough analysis of chromosomal changes
to obtain a comprehensive view of genomic alterations, both large and small (Table 1). Genomic approaches
can detect:
••
•
•
•
•
•
•
•
•

Amplifications and deletions
Loss of heterozygosity (LOH)
Uniparental disomy (UPD)
Balanced translocations or inversions
Unbalanced translocations
Mosaicism
Copy number variations (CNVs)
Single Nucleotide Variations (SNVs)
Small insertions and deletions
Structural variants

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

–3–

Table 1: Genomic Technologies for Cytogenetics

Parameter

FISH

Karyotyping

ArraysNGS(large gene panel)

NGS(whole genome)

Whole-Genome View

NoYes

Yes

No

Yes

Resolution> 50 kb> 5 Mb< 1 kb1 base pair1 base pair

Aneuploidy

Yes

Yes

Yes

Yes

Yes

Unbalanced
Translocation

Yes, if known

Yes, if large

Yes

Yesa

Yes

Balanced Translocation
or Inversion

Yes, if known

Yes

NoNoYesMosaicism

Yes

Yes

Yes, if 20% of cells
presentYes

Yes

Polyploidy

Yes (indirect)

Yes

Yes
(SNP arrays only)

Yes

Yes

UPDNo

No

Yes
(SNP arrays only)

Yesb

Yes

Copy-Neutral LOH

No

No

Yes
(SNP arrays only)

No

Yes

SNVs

No

No

No

Yes

Yes

Gene Fusions

Yes

NoYes, if unbalanced
No, if balanced

Yes

Yes

Using arrays and NGS to complement traditional methods, cytogeneticists can obtain a comprehensive view of genetic abnormalities, both large and small.2a. Yes, if branch points are targeted.
b. Yes, if both parents are analyzed.

“We are moving from cytogenetics to cytogenomics. It seems in the near future, array CGH andNGS will replace conventional cytogenetics in most clinical and research applications.”2

II. Genomic Technologies for Cytogenetics
Genomic technologies improve upon previous methods by allowing a wider view of the genome in a single assay.
Array and NGS applications offer advantages in resolution and scale for the detection of chromosomal abnormalities.

ArraysBoth FISH and array methods rely upon nucleic acid hybridization, with the use of designed probes to detect specific
DNA targets. However, arrays can probe thousands of genetic loci simultaneously, providing wider coverage of thegenome and higher throughput in initial stages of testing than FISH. Arrays can validate known abnormalities, such

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

–4–

as deletions or amplifications, previously identified using standard cytogenetics, and discover numerous abnormalities
that were below the resolution of other cytogenetic tests.8
Chromosomal abnormalities that don’t involve copy number variations, such as inversions and balanced
translocations, might not be detected by arrays measuring signal intensity only.8 Single nucleotide polymorphism(SNP) arrays combine intensity information with genotyping information to enable identification of abnormalities thatwould be missed by either parameter alone, such as uniparental disomy (UPD). Combining genotyping with intensity
information can also extract copy number information in a background of normal cells in a heterogenous population.
Intercellular heterogeneity, also referred to as mosaicism, is often found in tumor samples mixed with normal cells, aswell as some genetic disorders. Mosaicism can be detected on Infinium® SNP genotyping arrays (Figure 1), but might
not be correctly identified on array CGH platforms that provide only intensity information.9

Figure 1: Genome-Wide View of Chromosomal Alterations—Horizontal view of the entire genome from BlueFuse® Multi Software showing
mosaicism detection. By correlating the intensity information from the log R ratio (top plot) and the genotyping information from the B Allele
Frequency Chart (middle plot), a mosaic deletion is evidenced on the long arm of chromosome 5. A region demonstrating low-level mosaic loss
(highlighted yellow) lies adjacent to a region of high-level mosaic loss, which is indicated by the position of the heterozygous track on the B Allele
Frequency Chart. The mosaic is unlikely to have been correctly characterized on an array CGH platform, which provides intensity information only.
Data shown is from the Infinium CytoSNP-850K BeadChip.

“NGS enables the detection of unexpected aberrations, such as SNVs, that aren’t wellcharacterized. Pairing these 2 methods—dose analysis for CNVs and sequence analysis forSNVs—will determine the genomic basis for an increasing number of inherited disorders.”—Udo Koehler, PhD, Lab Director, MGZ Medical Genetics Center, Munich, Germany10

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

–5–

Next-Generation Sequencing
Arrays can be designed for wide coverage of the genome with specific applications. However, there are a few things
that they cannot detect, such as balanced rearrangements.7 Although array technologies can detect variations at highresolution, new sequence variations cannot be detected, as prior knowledge is required for assay design. NGS data
can complement array data by providing additional information on single nucleotide level sequence variations, andchromosomal abnormalities such as balanced translocations.11
NGS technology is fundamentally similar to traditional Sanger sequencing, resulting in single base resolution of DNAsamples. The critical difference is that, instead of sequencing a single DNA fragment, NGS extends this processacross millions of fragments in a massively parallel fashion, providing enhanced coverage of the genome andhigher throughput. As with arrays, NGS offers flexibility in its application. Cytogeneticists have the option of rapidly
sequencing the entire genome or deeply sequencing targeted regions.

III. What is Next-Generation Sequencing?
NGS is a high-throughput process for determining DNA sequence. Illumina sequencing by synthesis (SBS) chemistry
uses a DNA polymerase to catalyze the incorporation of fluorescently labeled nucleotides into a DNA template strand
during sequential cycles of DNA synthesis. During each cycle, the nucleotides are identified by fluorophore labels.
This method is highly scalable and can be applied to a subset of key genes or the entire genome.

The Basic NGS Workflow
All Illumina, NGS workflows include 4 basic steps.
1. Library Preparation. Sequencing library preparation begins by creating short DNA or cDNA fragments with 5' and
3' adapters ligated (Figure 2A).
2. Cluster Amplification. For cluster generation, the library is attached to an oligonucleotide lawn on the surface of aflow cell. Through bridge amplification, each library fragment acts as a seed to generate a clonal cluster containing
thousands of identical fragments. Across the entire flow cell, millions to billions of clusters are formed (Figure 2B).
3. Sequencing. Next, the templates are ready for sequencing. SBS technology uses a proprietary reversible
terminator-based method that detects single bases as they are incorporated into DNA template strands.12
Because all 4 reversible terminator bound dNTPs are present during each sequencing cycle, natural competition
minimizes incorporation bias and greatly reduces raw error rates compared to other technologies.13,14 The result
is highly accurate base-by-base sequencing that virtually eliminates sequence context-specific errors, even within
repetitive regions and homopolymers (Figure 2C).
4. Alignment and Data Analysis. Newly identified sequence reads are exported to an output file and aligned to areference genome by sequencing alignment software (Figure 2D).
For a detailed animation of SBS technology, visit www.illumina.com/SBSvideo.

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

–6–A. Library Preparation

B. Cluster Amplification

Genomic DNA
Fragmentation
Flow Cell

Adapters
CyclesLigation

Sequencing
Library1234

Clusters

NGS library is prepared by fragmenting a gDNA sample and ligating specialized
adapters to both fragment ends.

C. Sequencing

D. Alignment & Data Anaylsis

T2134GACReads

Sequencing Cycles

Data is exported to an output file

Digital Image

Reference
GenomeCluster 1 > Read 1: GAGT...
Cluster 2 > Read 2: TTGA...
Cluster 3 > Read 3: CTAG...
Cluster 4 > Read 4: ATAC...
Text File
Sequencing reagents, including fluorescently labeled nucleotides, are addedto the flow cell and the first base is incorporated. The flow cell is imaged and theemission from each cluster is recorded. The emission wavelength and intensity
are used to identify the base. This cycle is repeated “n” times to create a read
length of “n” bases.

Reads are aligned to a reference sequence with bioinformatics software. Afteralignment, differences between the reference genome and the newly sequenced
reads can be identified.

Figure 2: Next-Generation Sequencing (NGS) Workflow—The Illumina NGS workflow follows 4 basic steps: library preparation, cluster
generation, sequencing, and data alignment.

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

–7–Multiplexing
Over time, NGS has increased data output per run, while also increasing sample throughput. Multiplexing allows
large numbers or batches of libraries to be pooled and sequenced simultaneously during a single sequencing run(Figure 3). With multiplexing, unique index sequences are added to each DNA fragment during library preparation sothat each read can be identified and sorted before final data analysis. This dramatically reduces the time-to-data for
multisample studies and enables researchers to go from experiment to answer faster and easier than ever before.

CATTCGACGGATCGCATTCGTGGCAGTCCATTCGCAGTTCATT
CATTCGAACTTCGA

Sequence Output
to Data File

AACTGAGTCCGATA
AACTGATCGGATCCAACTGAACCTGATG
AACTGAGATTACAACATTCGACGGATCGAACTGAGTCCGATA
AACTGATCGGATCCCATTCGTGGCAGTCAACTGAACCTGATG
Library 1 Barcode

AACTGAGATTACAA

Library 2 Barcode

CATTCGCAGTTCATT

Sequencing Reads

CATTCGAACTTCGADNA Fragments
Reference Genome

Figure 3: Library Multiplexing Overview—A. Distinct samples are attached to unique index sequences. Index sequences are attached duringlibrary preparation. B. Libraries for each sample are pooled together and loaded into the same flow cell lane. C. Samples are sequenced together
during a single instrument run. All sequences are exported to a single output file. D. A demultiplexing algorithm sorts the reads into different files
according to their indexes. E. Each set of reads is aligned to the appropriate reference sequence.

IV. Summary
Over the last decade, advances in genomic technologies have led to improved approaches to characterizing genetic
abnormalities. In turn, improved analyses have increased understanding of genetic influences on many diseases.
With more thorough, accurate methods for detection of chromosomal aberrations, genomic technology offers new
possibilities for identifying and monitoring diseases in the future.
Illumina is committed to advancing molecular testing tools and collaborating with industry leaders to transform healthcare. Together, we aim to bring the promise of genomics toward widespread clinical adoption and improvements inpatient diagnosis, treatment, and outcomes.

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

–8–

V. Glossary
Adapters: Oligos bound to the 5' and 3' ends of each DNA fragment in a sequencing library. The adapters arecomplementary to the lawn of oligos present on the surface of Illumina sequencing flow cells.
Bridge amplification: An amplification reaction that occurs on the surface of an Illumina flow cell. During flow cellmanufacturing, the surface is coated with a lawn of 2 distinct oligonucleotides referred to as “p5” and “p7.” In the firststep of bridge amplification, a single-stranded sequencing library is injected into the flow cell. Individual moleculesin the library bind to complementary oligos as they “flow” across the oligo lawn. Priming occurs as the free end of aligated fragment bends over and “bridges” to another complementary oligo on the surface. Repeated denaturationand extension cycles (similar to PCR) result in localized amplification of single molecules into millions of unique, clonal
clusters across the flow cell.
Clusters: A clonal grouping of template DNA bound to the surface of a flow cell. Each DNA template strand thatbinds to the flow cell acts as a seed and is clonally amplified through bridge amplification until the cluster has roughly
1000 copies. Each cluster on the flow cell produces a single sequencing read. For example, 10,000 clusters on theflow cell would produce 10,000 single reads.
Flow cell: A glass slide coated with a lawn of surface-bound, adapter-complimentary oligos. A pool of8–384 multiplexed libraries can be sequenced simultaneously, depending on application parameters.
Indexes/Barcodes/Tags: A unique DNA sequence ligated to fragments within a sequencing library for downstreamin silico sorting and identification. Libraries with unique indexes can be pooled together, loaded into a lane of asequencing flow cell, and sequenced in the same run. Reads are later identified and sorted via software.
Multiplexing: Multiple samples, each with a unique index, can be pooled together, loaded into the same flow cell,and sequenced simultaneously during a single sequencing run. Depending on the application and the sequencing
instrument used, 10–384 samples can be pooled together.
Read: In general terms, a sequence “read” refers to the data string of “A, T, C, and G” bases corresponding to thesample DNA. With Illumina technology, millions of reads are generated in a single sequencing run. In specific terms,each cluster on the flow cell produces a single sequencing read.

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

–9–VI. References
1.

Morozova O, Marra MA. From cytogenetics to next-generation sequencing technologies: advances in the detection of genome rearrangements in tumors.
Biochem Cell Biol. 2008;86:81-91.
2. Halder A, Jain M. Cytogenetics to cytogenomics: transition from chromosome to DNA sequence. Global Journal of Human Genetics & Gene Therapy.
2014;2:53-67.
3. Petrone J (2010) ACMG Recommends Replacing Karyotyping with Chromosomal Microarrays as ‘First-Line’ Postnatal Test. Genomeweb (www.genomeweb.com/arrays/acmg-recommends-replacing-karyotyping-chromosomal-microarrays-first-line-postnat) Accessed 10 October 2015.
4. Jackson EM, Sievert AJ, Gai X, et al. Genomic analysis using high-density single nucleotide polymorphisms-based oligonucleotide arrays and multiplex
ligation-dependent probe amplification provides a comprehensive analysis of INI1/SMARCB1 in malignant rhabdoid tumors. Clin Cancer Res. 2009;15:1923-1930.
5. Ho CY, Mosier S, Safneck J, et al. Genetic profiling by single-nucleotide polymorphism-based array analysis defines three distinct subtypes of orbital meningioma.
Brain Pathol. 2015;25:193-201.
6. Harada S, Henderson LB, Eshleman JR, et al. Genomic changes in gliomas detected using single nucleotide polymorphism array in formalin-fixed,
paraffin-embedded tissue: superior results compared with microsatellite analysis. J Mol Diagn. 2011;13:541-548.
7. Illumina (2015) Microarray-based cytogenetic testing illuminates congenital abnormalities and cancer.
(www.illumina.com/content/dam/illumina-marketing/documents/icommunity/article_2015_01_combimatrix_cytosnp850.pdf)
8. Illumina (2010) Cytogenetics into Cytogenomics: SNP Arrays Expand the Screening Capabilities of Genetics Laboratories.
(www.illumina.com/documents/products/appnotes/appnote_cytogenetics.pdf)
9. Illumina (2012) Molecular Cytogenetics.
(www.illumina.com/documents/products/datasheets/datasheet_cytogenetics.pdf)
10. Illumina (2015) From arrays to NGS: how chromosomal genetics evolved from structure to disease.
(www.illumina.com/content/dam/illumina-marketing/documents/icommunity/koehler-mgz-cyto-article-1570-2015-008.pdf)
11. Liang D, Peng Y, Lv W, et al. Copy Number Variation Sequencing for Comprehensive Diagnosis of Chromosome Disease Syndromes. J Mol Diagn. 2014;16(5):519-526.
12. Bentley DR, Balasubramanian S, Swerdlow HP, et al. Accurate whole human genome sequencing using reversible terminator chemistry.
Nature. 2008;456(7218):53-59.
13. Ross MG, Russ C, Costello M, et al. Characterizing and measuring bias in sequence data. Gen Biol. 2013;14:R51.
14. CARDIoGRAMplusC4D Consortium. Large-scale association analysis identifies new risk loci for coronary artery disease. Nat Genet. 2012;45(1):25-33.

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

– 10 –

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.
© 2016 Illumina, Inc. All rights reserved. Illumina, BlueFuse, and the pumpkin orange color are trademarks of Illumina, Inc. and/or itsaffiliate(s) in the U.S. and/or other countries. Pub. No. 1170-2015-007 Current as of 13 March 2016