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Apr 1, 2006 (Vol. 26, No. 7)

Taming the Genome with Microarrays

We have the Technology and an Abundance of Information. What's Next?

  • When scientists finished mapping the human genome, they thought their work was done. However, they have since found out that there is such a thing as too much information.

    There are only a little more than a handful of companies that are pioneering different versions of microarray technology, but each has found its own niche in this rarified area of bioresearch.

  • Affymetrix

    Affymetrix’ (www.affymetrix.com) GeneChip platform includes oligonucleotide arrays, reagents to prepare samples for hybridization to arrays, instruments to process and analyze the arrays, and software tools to manage and mine the data.

    Current Affymetrix arrays have more than six million data points, compared with 16,000 on the first commercial array produced in 1994, thus helping scientists perform experiments that were not possible before. The GeneChip expression arrays enable researchers to simultaneously monitor genome-wide expression profiles to understand biological mechanisms of complex diseases and processes and identify new drug targets in new ways. Customers can select from available catalog arrays or design their own arrays.

    Affymetrix recently expanded its gene-expression product line with whole-genome tiling arrays that interrogate all of the nonrepetitive sequence of a genome. The company has introduced whole-genome human and mouse tiling arrays and will be launching additional tiling arrays for Arabisopsis, S.cerevisiae, and S.pombe in the near future. Each array generates almost 6.5 million data points at regular 35 base pair intervals across the entire genome.

    Affymetrix DNA analysis technology enables scientists to look at as many as 500,000 SNPs throughout the entire human genome, according to the company. Each SNP on a GeneChip Human Mapping Array is interrogated with approximately 40 different probes. The Human Mapping 500K Array Set enables researchers to perform whole-genome association studies across tens of thousands of samples. They can use the 500K to search the entire genome and then use targeted genotyping assays to follow up on their genes and SNPs of choice, all on one platform.

  • NimbleGen Systems

    NimbleGen Systems (www.nimblegen.com) manufactures high-density arrays of long isothermal probes using Maskless Array Synthesis technology. One application, Comparative Genomic Hybridization (CGH), measures DNA copy-number differences between a reference genome and a researcher’s sample genome.

    NimbleGen offers two high-definition array CGH products; whole-genome array CGH and fine-tiling array CGH. Whole-genome CGH measures copy-number differences in DNA across entire genomes, while fine-tiling CGH determines breakpoints at ultrahigh resolution in DNA targets located in any subset of DNA, either a contiguous region or many different regions of interest.

    “Customers are excited by the combination of high-quality and high-resolution data that we provide from a single CGH experiment,“ asserts Emile Nuwaysir, vp of business development. “Our CGH arrays are already the highest resolution products available, and our next-generation products will be even better.“

    Key features of NimbleGen’s microarrays, such as ultrahigh density and long oligo probes, yield a powerful, high-definition CGH platform for whole-genome analysis, according to the company. With the flexibility of NimbleGen’s array synthesis, chromosomal aberrations identified in whole-genome surveys can be readily examined at even higher resolution with custom fine-tiling array CGH.

    Such studies can map genomic changes down to the gene, as well as exon level and enable breakpoint mapping to less than 500-bp intervals. NimbleGen’s array CGH detects both single and multiple copy changes.

    NimbleGen’s high-capacity microarrays contain 385,000 probes on a single glass slide with next-generation prototypes containing more than four million probes. The current density enables design of a human whole-genome CGH array with probes tiled through genic and intergenic regions at a median probe spacing of 6,000 bp.

    This tiling-path array-design approach ensures researchers get the complete picture on genome-wide copy-number changes. Ultrahigh resolution detection of small deletions and amplifications, as well as breakpoint mapping to less than 500-bp intervals can be achieved using NimbleGen’s fine-tiling arrays with probes as dense as 10-bp spacing.

    NimbleGen’s exclusive isothermal array-design approach enables uniform probe performance, eliminating hybridization artifacts and/or bias and providing higher quality data, the company claims. Probe lengths are adjusted (45󈟁 mer) to equalize the melting temperature (Tm = 76C) across the entire set. Thus probes are optimized to perform equivalently at a given stringency in specific genomic regions, including AT- and GC-rich regions.

    “A large market has emerged for the use of microarrays in novel applications, such as CGH, ChIP-chip, and DNA methylation,“ Nuwaysir adds. “Traditional gene-expression microarray experiments offer a limited view of genome biology. To see the whole picture, a multidimensional approach is required that tests for gene expression, as well as DNA copy number, DNA methylation, histone code, transcription factor binding, and SNP genotyping.“

  • Illumina

    Illumina (www.illumina.com) developed a range of array-based offerings for scanning and analysis on the BeadStation system that also support automation and LIMS options that enable customers to acquire end-to-end genetic analysis capability.

    Illumina offers two complementary approaches to array content design; gene-specific probes and universal probes. It also synthesizes and makes available a range of reagents and assay kits for use with its arrays. Illumina arrays support gene-expression profiling and SNP genotyping.

    “For key study areas and model organisms, Illumina continues to develop standard catalog expression arrays,“ says Todd Dickinson, associate director of product marketing. “For focused approaches, we can deploy gene-specific probe content on either of two multisample Sentrix Array platforms—the Sentrix Array Matrix and the Sentrix BeadChip.

    “Genome-wide probe content (23 K� K probe sequences/ sample) is available only on our BeadChips, which can be configured to contain over 12 million, three-micron features. For instance, our whole-genome expression chips allow customers to process six samples, 48,000 transcripts a piece on one BeadChip. We’ve also developed our novel DASL assay for reproducible expression profiling of degraded RNAs, such as those found in FFPE samples. This opens the door for biomarker validation or retrospective studies.“

    For genotyping, Illumina has developed the high-multiplex GoldenGate assay protocol for preparation, labeling, and amplification of sample target sequences prior to hybridization to the universal address sequences on the 96-sample Sentrix Array Matrix or the Sentrix-16 BeadChip.

    “The GoldenGate protocol became the gold standard of the HapMap Project,“ according to Sarah Shaw Murray, Ph.D., Illumina staff genotyping science manager. “Early this year, Illumina began shipping the first true post-HapMap array product, the HumanHap300 BeadChip. With tagSNP-centric content and a new assay, Infinium, our new HumanHap BeadChips deliver best-in-class genomic coverage and data quality for whole-genome disease association studies—the fastest-growing microarray market segment.“

  • Applied Biosystems

    Applied Biosystems(www.appliedbiosystems.com) offers a comprehensive range of DNA analyzers for applications, such as sequencing, resequencing, and SNP genotyping. Companion products, such as the VariantSEQr Resequencing System and the SNPlex Genotyping System, integrate all necessary components, including assays and software, to conduct specific applications.

    The company also offers an integrated workflow solution for gene-expression analysis to enable researchers to move from whole-genome analysis using the Expression Array System to gene quantification and single-gene analysis with Applied Biosystems’ TaqMan Gene Expression Assays and TaqMan Low Density Array real-time PCR systems, as well as Celera Discovery System Online Platform for protein identification.

    Applied Biosystems offers a selection of genotyping products, including the SNPlex Genotyping System for flexible, high-throughput analysis, and approximately two million ready-to-use human SNP assays. They also offer complete solutions in instrumentation and tagging chemistries for protein identification and quantification.

    ICAT reagents are a protein-based labeling strategy when comparing two or more samples, while new iTRAQ reagents allow researchers to multiplex up to four different samples, providing broad protein coverage, including information on post-translationally modified peptides.

    “One of the biggest bottlenecks we’ve come up against is data analysis, particularly for new microarray researchers,“ says Christopher Streck, product manager. “To that end we provide Web tools to place the capabilities in users’ hands.“

    “Outsourcing is one of the biggest trends we are seeing,“ adds Roland Wicki. “A lot of our customers come from the real-time space and need services as part of our service provider program. We also have a separate integration program to help integrate AB products as well as third-party and in-house instruments and software to develop customized, workflow-specific solutions.“

  • Premier Biosoft

    “Premier Biosoft International (www.premierbiosoft.com) offers software products to empower the researcher,“ says Kay Brown, president and CEO. “With AlleleID, the researcher can not only design thousands of specific, efficient oligos for spotting on microarrays but can even verify the results using quantitative PCR (qPCR).

    “We offer the choice of designing end-point detection SYBR Green primers or dual-labeled probes for TaqMan, FRET, or molecular beacon assays. Our users report that the designs consistently perform well in the labs. We think that this assay success is attributable to several unique features built into the program. The program interprets BLAST results to assure oligo specificity, folds the templates to assure efficiency, and a proprietary algorithm uses statistical optimization techniques to design the best oligos.

    “With ClustalW multiple sequence alignment at its core, AlleleID can design microarrays or qPCR assays to distinguish any branch or leaf of a philogenic tree. For example, you can design assays for distinguishing E. coli strains or species,“ says Brown.

    “Or you can use the capability for cross-species work to apply the understanding of model organisms to species of interest,“ she continues. “You can develop diagnostic assays for pathogen detection, bacterial identification, environmental monitoring, or even for infectious diseases. For developing strain identification assays, it is customary to design one set of oligos for each strain. AlleleID incorporates a unique minimal-set capability to save assay costs, made possible by an innovative algorithm, developed by our team of statisticians.“

    In this post-sequencing era, studying just gene-expression patterns is deemed inadequate. Scientists now want to study in detail the control elements of genes, epigenetic modifications, alternate splicing, and many other aspects of how biological diversity is controlled by the DNA molecule. Array Designer, the company’s microarray design product, offers tiling array and resequencing array capabilities.

    One can design a tiling array with as low as one base resolution to look at base-by-base effects. Resequencing arrays are a newer technique for population studies, because hybridization sequencing is rapid, accurate, and far less expensive than capillary or dideoxysequencing.

    “We have a slightly different approach; we are all about enabling scientists to do their research,“ Brown asserts.

  • GE Healthcare

    For GE Healthcare (www.gehealthcare.com), it’s all about statistical relevance, says John Burczak, head of R&D. “You need to know whether the differences you find in gene expression are statistically relevant. To that end, we provide products for sample processing and data analysis in addition to our microarray offerings.“

    In general, GE Healthcare’s customers are asking the following questions: How does a particular gene function? Can they identify differences in gene expression between cell types? And what insight can they gain into molecular processes?

    The CodeLink whole-genome array series offers researchers the most comprehensive expression data commercially available from a single slide, according to the company. The platform’s sensitivity, reproducibility, specificity, and accuracy allow researchers to detect more genes, particularly low-abundance genes, consistently and accurately.

    “Customers are looking at the cascade of events that happens with regard to gene function, finding differences between cell types, and the molecular processes that drive the cell,“ says Burczak. “In the near term, their biggest problem in the microarray arena is getting the data-analysis tools to give them the answers they are looking for.“

    Not that there’s any shortages of information. “You generate lots of data that needs analysis,“ adds Burczak. “And what we’re doing to solve the problem is to develop software with an easy user interface to analyze data for statistically relevant information.“

    Burczak also sees a trend toward industry standardization. “Manufacturers are moving closer to matching standards for their individual platforms. We are seeing a convergence—today maybe 80% of data from different microarray platforms matches. We’ll get closer to 100% matching between platforms as the technology moves forward.“

    For GE Healthcare (www. gehealthcare. com), it’s all about statistical relevance, says John Burczak, head of R&D. “You need to know whether the differences you find in gene expression are statistically relevant. To that end, we provide products for sample processing and data analysis in addition to our microarray offerings.“

    In general, GE Healthcare’s customers are asking the following questions: How does a particular gene function? Can they identify differences in gene expression between cell types? And what insight can they gain into molecular processes?

    The CodeLink whole-genome array series offers researchers the most comprehensive expression data commercially available from a single slide, according to the company. The platform’s sensitivity, reproducibility, specificity, and accuracy allow researchers to detect more genes, particularly low-abundance genes, consistently and accurately.

    “Customers are looking at the cascade of events that happens with regard to gene function, finding differences between cell types, and the molecular processes that drive the cell,“ says Burczak. “In the near term, their biggest problem in the microarray arena is getting the data-analysis tools to give them the answers they are looking for.“

    Not that there’s any shortages of information. “You generate lots of data that needs analysis,“ adds Burczak. “And what we’re doing to solve the problem is to develop software with an easy user interface to analyze data for statistically relevant information.“

    Burczak also sees a trend toward industry standardization. “Manufacturers are moving closer to matching standards for their individual platforms. We are seeing a convergence—today maybe 80% of data from different microarray platforms matches. We’ll get closer to 100% matching between platforms as the technology moves forward.“

  • Agilent Technologies

    Agilent (www.agilent.com) custom-designed microarrays enable researchers to apply ChIP-on-chip technology tailored to their area of research.

    “There are only so many organisms out there,“ notes Scott Harrison, product manager. “Whether we’re talking about model organisms or unique organisms, the biology community now has the tools available. Sequencing has gotten very inexpensive.“

    Agilent supplies a broad custom content from their tiled whole-genome databases for a large selection of organisms, including human, mouse, rat, yeast, Arabidopsis, and C. elegans. There is greater confidence in binding event data. Powerful algorithms employ neighborhood probe voting with multiple probes to generate reliable data with greater true binding events and fewer false positives.

    “The inherent flexibility of our microarrays allows users to define content—up to 44K customizable features per microarray—for any tiling density, genomic region, and organism of interest,“ explains Harrison. “But the real core part of our business is our flexible printing capability.“

    Agilent’s SurePrint microarray technology features a flexible, industrial-scale inkjet printing process that synthesizes oligonucleotide probes in situ onto 1“ x 3“ slides. The maskless process allows quick iteration of microarray designs in response to fast-paced content changes in the continuously evolving genomics environment. This allows researchers access to high-quality arrays loaded with relevant and rich content.

    ChIP Analytics Software, Agilent’s data analysis tool, combines annotated, algorithmic array data processing with an easily manipulated text-file output and high-speed statistical modeling functions. In addition, access to probe sequences and annotation files to facilitate searching of public databases and extraction of biological information is provided through Agilent’s eArray application.

  • Ocimum Biosolutions

    Ocimum Biosolutions (www.ocimumbio.com) provides an integrated while open approach for microarray solutions that includes catalog and custom arrays, oligo sets, hybridization services, as well as software and data-analysis services. This enables customers to either completely outsource their microarray experiments or select one of the products or services.

    Ocimum supplies an array technology that provides reproducible, easy-to-evaluate results. “All our developments are made from a customer’s perspective and aim to make experimental procedures as simple as possible without compromising on quality,“ notes Christian Betz, global head of production.

    Ocimum’s approach allows complete control and flexibility over the design and production, avoids contamination, eliminates risk of cross-hybridization, reduces background signals, and gives high-reproducibility, according to the company.

    “We have a very integrated approach, as not many can provide an end-to-end solution to their customers,“ says Anuradha Acharya, CEO. “Our position is also strong in custom arrays. We have a lot of experience in creating custom arrays for customers worldwide and it will continue to be one of our strong points while we continue to provide many more catalog arrays.“

    Ocimum took over MWG’s (www.mwgbiotech.com) portfolio of biochips and rebranded them as OciChipsT. These include microarrays and oligo sets, covering complete genomes of a multitude of model organisms, including E. coli, yeast, rat, mouse, zebrafish, and Arabidopis, as well as a biochip representing the complete human genome.

    “We have identified new plant and animal genomes to be added this year. We have already added the new rat 30 K OciChip and have done quite a few custom arrays,“ Acharya adds.

    “Our software and data-analysis services include Oligostar for Oligo design, a package called Genowiz for data analysis, and the Biotracker LIMS. Both Genowiz and Biotracker are Affymetrix GeneChip compatible. Not only that, we also allow researchers to compare across different platforms and experiments using our software products,“ says Acharya.

  • The Road Ahead

    As divergent as these technologies are, the field appears to be moving toward convergence and standardization. “The trends I see in the field are smaller customized arrays (topic arrays) rather than genome-wide arrays, lower prices, and outsourcing of experiments,“ says Betz.

    “In terms of diagnostics, traditional methods like ELISA and RT-PCR will get competition from specialized microarrays. There will be a battle between multiplex RT-PCR and small microarrays. And in terms of detection long-term, there will be a merging of the electronic chip technology and the biochip to allow online detection via changes in electric parameters, avoiding the need for fluorescent or other labeling.“



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