Data Sheet: DNA Analysis

Molecular CytogeneticsIllumina has a broad portfolio of products—from microarrays to massively parallel sequencing—for identifying structural variants and studying their role in human disease.

Figure 1: Illumina Beadarray® and Sequencing Platforms

The Infinium® HD Assay is deployed on multi-sample BeadChip microarrays and read with the iScanTM Reader (left). Illumina sequencing technology is performed on an eight-lane flow cell in the Genome Analyzer (right).

Illumina Cytogenetics Tools Highlights

• High-Quality Data: Low noise on arrays and essentially no noise with sequencing for sensitive detection of copy number changes

• Unsurpassed Flexibility: Reliable detection of deletions, duplications, inversions, rearrangements, and amplifications

• High Resolution: BeadChips with down to 1.5kb marker spacing, and sequencing for single-base resolution

• Easy Workflows: Fast, simple, and proven sample preparation protocols, automated processing, and data readout

• Software Support: Suite of analysis and visualization programs with an open architecture to support integration with other downstream analysis tools

Structural Variation and PhenotypeIn addition to SNPs, structural variability is now recognized as a substantial source of genetic variation that has a major influence on phenotypic variation. Thus, genome-wide profiling for chromosomal aberrations—such as amplifications, deletions, and rearrangements—is crucial for both the study of cancer and congenital disorders. Recently, the study of DNA copy number variation has become an essential component of whole-genome disease association studies1-5.

Cytogenetics, the study of chromosomes and their abnormalities, has a history of evolving tools permitting increasingly fine-grained profiling of genomes. The field began in the 1960s, when chromosomes were analyzed via G-banding. In the 1980s, molecular cytogenetics meth-ods such as fluorescent in situ hybridization (FISH) began to be used widely. In the 1990s comparative genomic hybridization (CGH) was developed, and eventually higher-density CGH was implemented on microarrays (array-CGH).

Illumina has developed a portfolio of the next generation of modern genomic analysis tools for identifying and measuring these impor-tant variants, including point mutations, copy number changes, and rearrangements (Figure 1). Recent advances in SNP genotyping array technology have enabled Illumina DNA Analysis BeadChips to profile structural variation with nearly 1.2 million markers. In addition, now Illumina high-throughput sequencing allows the analysis of any type of structural variant at single base-pair resolution6,7.

Profiling Structural Aberations Illumina analysis products can detect all classes and all sizes of struc-tural variation in the human genome that affect phenotypes (Table 1).

Duplications, Deletions, and AmplificationsIllumina DNA Analysis BeadChip data provide information about changes in copy number, due to signatures in probe signal intensities and allelic ratios. Thus, deletions (reduction in copy number) and am-plifications (increase in copy number) are readily identified by analyzing Illumina BeadChip data for these signature profiles (Figure 5A-C).

RearrangementsIllumina sequencing identifies rearrangements, in addition to copy number changes and point mutations, at single base pair resolution. Paired-end sequencing accurately aligns fragments to a region of the genome, even when it is in an unexpected location relative to a refer-ence genome.

Copy-Neutral LOH The unique ability of SNP genotyping arrays to simultaneously mea-sure intensity differences and allelic ratios allows for the profiling of both DNA copy number and copy-neutral LOH (Figure 5D). Copy-neutral LOH is where loss of heterozygosity occurs with no physical change in the copy number of a particular region. In tumorigenesis, copy-neutral LOH is thought to arise from mitotic recombination, caused by a rare crossover event during mitotic cell division. The prod-ucts of mitotic recombination are regions of the genome exhibiting uni-parental disomy (UPD), in which both genomic regions originate from the same parent8. Only arrays that provide SNP genotyping informa-tion can detect copy-neutral events such as uniparental disomy (UPD), mitotic recombination, or gene conversion events. UPD has already

Data Sheet: DNA Analysis

Figure 3: High Signal-to-Noise Genotype Information

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SNP genotyping data (purple) provides an increased signal-to-noise ratio for detecting some aberrations, compared to using intensity data alone (green).

Figure 1: Illumina DNA Analysis and Sequencing Products Identify Structural Variation

Figure 2: SNP Array Detection of Mosaic Deletion

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The loss of intensity in log R ratio (top plot) indicates a deletion on chromo-some 3 in this tumor sample. The genotyping information (bottom plot) shows evidence that this is a mosaic sample. The position of the two central populations of data points (at 0.7 and 0.3), imply that this sample contains 70% normal cells and 30% tumor cells. This mosaic would not have been correctly identified on an array-CGH platform, which provides only intensity information.

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Adapted from Speicher and Carter, 2005

been linked to such disorders as Prader-Willi Syndrome and Angelman Syndrome9. Studies using more powerful tools are expected to show that copy-neutral events play a much larger role in tumorigenesis than is currently appreciated10.

Mosaicism The use of SNP arrays for general screening of common aberrations is relatively straightforward. However, results can be confounded by intercellular heterogeneity (also referred to as mosaicism), contamina-tion from normal tissue when analyzing a tumor, or other complex patterns. Because aberrations may not occur in every cell and certain cells may have a normal karyotype while others contain aberrations,

identifying mosaics can be labor intensive. Arrays that provide both genotype and intensity information can extract copy number informa-tion in a background of normal cells or in a heterogeneous population of cells (Figure 2). With Illumina SNP arrays, genotyping information in the form of B allele frequency (an allelic composition measurement) can resolve mosaic samples. Using both the log R ratio (based on in-tensity information) and the B allele frequency parameters can reliably demonstrate the overall level of mosaicism and provide insight into the mechanism behind the mosaicism.

Data Sheet: DNA Analysis

Powerful Cytogenetics PortfolioIllu mina offers a wide spectrum of products on two different platforms to cater to cytogenetics research. Each platform provides important benefits, from the efficient and easy-to-run Infinium BeadChips to the ultimate resolution of high-throughput sequencing. Both platforms provide substantially better resolution to detect smaller regions than FISH or CGH. Illumina Infinium DNA Analysis BeadChips are easier to use and more efficient than array-CGH whole-genome analysis and provide high-density genotyping and CNV detection. The Infinium As-say supports essentially unlimited loci multiplexing and marker design, and underlies the industry’s most powerful structural variant detection panels (Table 2).

Illumina sequencing technology is the ultimate molecular karyotype and generates gigabases of sequence data per week, to quickly provide sufficient genomic coverage for the study of nearly all point mutations, structural variants, and indels, with precise breakpoint identification.

Efficient Screening of VariantsIllumina DNA Analysis BeadChips offer researchers unmatched analy-sis of structural variation integrated with industry-best coverage for powerful whole-genome SNP genotyping. All Infinium HD BeadChips are in a multi-sample format and can be combined with robotic au-tomation for highly efficient processing. Data generated with Illumina BeadChips can be used to characterize common congenital disorders and profile reported CNV regions while at the same time providing dense coverage for novel CNV discovery.

The Power of SNPsA majority of the markers on Illumina whole-genome DNA Analysis BeadChips consist of SNP genotyping probes. Using SNP genotyping data offers significant advantages for cytogenetic analysis. Since gen-otypes are essentially digital (allele A or allele B), the data have much higher signal-to-noise ratios (SNR) than if analysis depends exclusively on comparisons between signal intensities. For example, using the In-finium HD Assay (Figure 3), the information derived from the genotypes (B allele frequency) can detect a hemizygous deletion with a SNR of 9 (using a single SNP window). The intensity information alone (log R ratio) provides a SNR of 2 for detecting the same deletion (also using a single SNP window) and is only improved to a SNR of 5 when using a 10-SNP window11. Illumina’s robust clustering process and stable Infinium HD reagent configuration enables precise profiling of

copy number changes with genotype information. Furthermore, certain types of structural lesions such as copy-neutral LOH, UPD, mitotic recombination, and gene conversion are only detectable by using SNP genotyping, not array-CGH. Mosaicism is also amenable to study us-ing the SNP markers on Illumina BeadChips because of the combined use of genotyping information and intensity data.

High Density ArraysIllumina scientists have taken advantage of unconstrained marker selection to intelligently fill in gaps and provide uniform coverage across the entire genome. Infinium HD BeadChips feature up to 1.2 million SNPs and probes, with median marker spacing down to 1.5kb and dense coverage of the Database of Genomic Variants12 and the most common congenital disorders. The high density of markers on the DNA Analysis BeadChips allows precise determination of CNV breakpoints across multiple individu-als10,13-15.

Table 2: High Resoluction and Coverage with Illumina Products

Illumina Infinium HD Illumina Sequencing CGH BAC Arrays

GenomicCoverage(Mean)

CEU: 0.86-0.96 CHB+JPT: 0.82-0.95 YRI: 0.60-0.86

Complete n/a

(no available genotypes)

n/a (no available genotypes)

MarkerSpading

1.5kb-7kb Single-base 6.4kb-100kb 80kb-100kb

EffectiveResolution

10kb-50kb Single-based 60kb-1000kb 80kb-100kb

Data Sheet: DNA Analysis

CNV-Targeted ContentIn addition to the uniform genome-wide panel of markers, a set of ~60,000 markers, developed in collaboration with deCODE genetics for enhanced CNV detection16, are available on Infinium HD Bead-Chips. These markers target CNVs described in the Toronto Database of Genomic Variants12 as well as regions likely to contain novel CNVs, such as segmental duplications, megasatellites, and SNP deserts.

Custom-Designed ContentWith assistance from Illumina scientists and the proprietary Assay Design Tool, researchers can design a panel of up to 60,800 SNPs for a fully custom iSelect® 12-sample BeadChip or in addition to standard content panels. Custom-designed content can be added to the broad genome-wide standard content on the Human1M-Duo and Hu-man610-Quad BeadChips. The results are semi-custom Human1M-Duo+ and HumanHap550-Quad+ DNA Analysis BeadChips.

Infinium AssayThe Infinium Assay protocol (Figure 4) features single-tube sample preparation and whole-genome amplification without PCR or liga-tion steps, significantly reducing labor and sample handling errors. After hybridizing unlabeled DNA sample to the BeadChip, two-step allele detection provides high call rates and accuracy. Selectivity and specificity are accomplished in two steps. Target hybridization to bead-bound 50mer oligos provides high selectivity and the enzymatic single-base extension provides powerful specificity. The single-base extension also incorporates a labeled nucleotide for assay readout. Intensity and allele data are used to determine regions of structural aberration.

The Infinium Assay allows nearly unlimited freedom to design mark-ers at rationally selected loci. This flexibility has permitted Illumina scientists to create the most powerful marker panels for both SNP genotyping and identification of structural variants. Markers on Infinium DNA Analysis products are present at an average 18-fold redundancy. This feature redundancy lends to the outstanding data quality and low noise that are hallmarks of Illumina BeadChips. Low overall noise levels allow reliable detection of single changes in copy number and accurate breakpoint identification11.

Simplified Kit ConfigurationsTo further streamline the workflow, Infinium products are sold as kits that include BeadChips and all reagents required for processing. Kit sizes range from 16 to 384 samples to support efficient use in labs or study designs of all sizes and throughput needs.

Ultimate Molecular KaryotypeWhereas Illumina DNA Analysis BeadChips provide the easiest and most efficient method for focused analysis of most structural aber-rations, sequencing with the Genome Analyzer provides the most complete and highest resolution digital karyotype for studying essen-tially any class of variant. The Genome Analyzer leverages innovative clonal template cluster generation and novel reversible terminator technology for massively parallel sequencing and determination of gigabases of highly accurate sequence data per week. Resequencing using Illumina’s paired-end methodology provides a whole-genome

Figure 4: Infinium Assay Workflow

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Data Sheet: DNA Analysis

sequence data set that can be compared to a reference and analyzed for variation ranging from SNP calling to classification of larger and more complex variants including deletions, translocations, inversions, and rearrangements. Since there are no probes to design, essentially any organism is amenable to study using Illumina sequencing.

Single-Base ResolutionIllumina sequencing can be used to study any variant in any se-quenced genome. With the paired-end strategy, base pair level acquired deletions, tandem duplications, inverted duplications, rear-rangements, and combinations of aberrations, can all be studied on one platform. Important for identifying fusion genes, balanced translo-cations and inversions can also be detected.

Using small insert sizes of 200–500bp, most variants can also be studied and easily confirmed via PCR. Samples with longer inserts can be incorporated to improve detection of larger aberrant regions. Single-base resolution allows for efficient detection of indels and microdeletions. This approach also allows extremely precise refining of breakpoints (Figure 7).

Scalable Read DepthIn addition to providing the ultimate data set for studying structural variants, Illumina sequencing technology offers several other unique features important for cytogenetics. Since sequencing data are base calls and are effectively digital, noise doesn’t negatively impact analysis to a great degree. Likewise, read depth is easily scaled in a way not possible for array data. For example, to provide more power to analyze heterogeneous samples or resolve very high copy numbers, researchers can collect more reads from additional lanes and combine data sets. Measurements well above 20 copies per locus have been described with this technique6.

Easy Sample PreparationThe Genome Analyzer System offers the simplest and most elegant workflow among ultra-high-throughput sequencing technologies. The unique sample preparation protocol uses standard molecular biology techniques—not emulsion PCR—and minimizes handling errors and contamination concerns, eliminating the need for robotics or clean

Figure 5: Examples of Structural Aberations Discovered with Illumina Beadchips

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In combination, log R ratio (top plots, red line represents smoothed average) and B allele frequency (bottom plots) parameters exhibit signature profiles that specifi-cally identify structural variants.

A: Amplifications B: Hemizygous Deletion

C: DUPLICATION D: COPY-NEUTRAL LOH

Data Sheet: DNA Analysis

rooms. Paired-end sample libraries are prepared in a straightforward one-day process. The fast workflow maximizes the capacity of the Genome Analyzer, and walk-away automation reduces overall project time and cost. The system is designed to fit in any lab, from smaller individual researcher labs to core labs or genome centers.

Analysis SoftwareBeadStudio software enables effective scanning of data generated from Illumina BeadChips for structural aberrations and copy num-ber variants. Illumina has developed several algorithms for detecting structural variants, and provides these as BeadStudio plug-ins. These fast and easy-to-use plug-ins provide copy number estimates with confidence scores and generate visual bookmarks on an integrated genome viewer (Figure 5) and chromosome browser. All of the results can easily be exported as cytogenetic reports for downstream analy-sis.

Illumina Pipeline software generates sequence data and aligned reads from the primary imaging output of the Genome Analyzer. The pipeline is a collection of customizable scripts in an open architecture that supports the use of downstream third-party informatics tools for SNP calling or detection of structural variants6,17.

Additionally, illumina•Connect18 is a collaborative program that facilitates the development of new third-party tools and applications for downstream analysis of data generated using Illumina products. illumina•Connect participants include several current providers of commonly used cytogenetics software.

Figure 6: Illumina Sequencing Detects Structural variants

Reference Genome

Sample Sequence

InsertionNormally Mapped Pair Deletion Inversion

Illumina Paired-End Sequencing can identify essentially any structural variation across the genome at single-base resolution. Paired reads (green arrows) are expected to have opposite orientation and to align with a uniform separation (defined by sample preparation). A specific variant is indicated when these expectations are violated in specific ways. Adapted from Korbel et al., 2007.

Figure 7: Breakpoint Detection with Illumina Paired-End Sequencing

Illumina Genome Analyzer Pipeline software includes tools to identify pairs of reads that may represent a structural variant. Pairs of reads in purple are farther apart than would be predicted from the uniform insert size (green), and identify a likely deletion.

ConclusionIllumina provides a broad portfolio of analysis tools for identifying point mutations, genomic rearrangements, and copy number changes of any size. Infinium array-based BeadChips are an efficient platform for whole-genome scanning and identification of many classes of struc-tural variation and copy number changes. These BeadChips make use of SNP genotype information along with probe intensity to generate higher signal-to-noise ratios for highly accurate discovery and identifi-cation even of relatively minor variants. Illumina sequencing leads the industry with the easiest sample preparation and massive sequencing throughput. This technology makes sequencing an affordable method to generate a single-base resolution molecular karyotype and analyze all classes of structural variation and SNPs.

Illumina, Inc. • 9885 Towne Centre Drive, San Diego, CA 92121 USA • 1.800.809.4566 toll-free • 1.858.202.4566 tel • techsupport@illumina.com • illumina.com

For research use only

© 2010 Illumina, Inc. All rights reserved.Illumina, illuminaDx, Solexa, Making Sense Out of Life, Oligator, Sentrix, GoldenGate, GoldenGate Indexing, DASL, BeadArray, Array of Arrays, Infinium, BeadXpress, VeraCode, IntelliHyb, iSelect, CSPro, GenomeStudio, Genetic Energy, HiSeq, and HiScan are registered trademarks or trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners. Pub. No. 370-2008-013 Current as of 28 July 2010

Data Sheet: DNA Analysis

Ordering Information

Product Catalog No.

Infinium HD BeadChips

HumanCNV370-Quad v3 BeadChips and reagents for processing 16 samples (4-pack) WG-311-1401

Human610-Quad v1 BeadChips and reagents for processing 16 samples (4-pack) WG-311-1001

Human1M-Duo v3 BeadChips and reagents for processing 16 samples (8-pack) WG-311-1105

Illumina Sequencing

Genome Analyzer SY-301-1201

Cluster Station SY-301-2001

Paired-End Module SY-301-1002

Paired-End Sample Prep Kit (10 samples) PE-102-1001

Paired-End Cluster Generation Kit (8 samples) GD-203-1001

36-cycle Illumina Sequencing Kit (8 samples) FC-104-1003

Representative kits shown. Other kit sizes are available and are listed in the Illumina Product Guide.

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3. Locke DP, Sharp AJ, McCarroll SA, McGrath SD, Newman TL, et al. (2006)

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4. Redon R, Ishikawa S, Fitch KR, Feuk L, Perry GH, et al. (2006) Global Varia-

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ping Translocation Breakpoints by Next-Generation Sequencing. Genome

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8. Ledbetter DH, Engel E (1995) Uniparental Disomy in Humans: Development of

an Imprinting Map and Its Implications for Prenatal Diagnosis. Hum Mol Genet

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Structural Genomic Variation in Ischemic Stroke. Neurogenetics Feb 21:

[Epub ahead of print].

11. Peiffer DA, Le JM, Steemers FJ, Chang W, Jenniges T, et al. (2006) High-

Resolution Genomic Profiling of Chromosomal Aberrations Using Infinium

Whole-Genome Genotyping. Genome Res 16: 1136-1148.

12. Database of Genomic Variants. http://projects.tcag.ca/variation (Build 36).

13. Stark M, Hayward N (2007) Genome-Wide Loss of Heterozygosity and Copy

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morphism Arrays. Cancer Res 67: 2632-2642.

14. Wang K, Li M, Hadley D, Liu R, Glessner J, et al. (2007) Penncnv: An Inte-

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15. Blauw HM, Veldink JH, van Es MA, van Vught PW, Saris CG, et al. (2008)

Copy-Number Variation in Sporadic Amyotrophic Lateral Sclerosis: A

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16. Expanding CNV Detection into the UnSNPable Genome Technical Note.

http://www.illumina.com/downloads/CNVdeCode_TechNote.pdf

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com/downloads/GASoftware_DS.pdf

18. Illumina•Connect. http://www.illumina.com/IlluminaConnect

Additional InformationTo learn more about Illumina cytogenetics analysis tools, DNA Analysis BeadChips, and sequencing, visit www.illumina.com or contact us at the address below.