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Mar 1, 2010 (Vol. 30, No. 5)

Targeted Resequencing Improves Precision

Ability to Focus on Specific Regions of Interest Pushes Technology to the Next Level

  • Custom Arrays

    According to Fred P. Ernani, Ph.D., senior product manager for emerging genomics applications at Agilent Technologies, “Agilent’s SureSelect Target Enrichment System is unlike any other commercially available target-enrichment method.” The platform is based on targeting with a biotinylated RNA library, which is hybridized with a genomic DNA sample to retrieve coding regions (the exome), which can then be bound to streptavidin-coated magnetic beads. “This approach allows us to build a custom array for the individual researchers’ needs.”

    The custom design of the system permits customers to expand their focus on specific genome regions to elaborate complex designs, including unusual organisms and specialized cell types, such as cancer cells and samples expressing rare human genetic mutations. Investigations of genetic diversity are a critical area of evolutionary and environmental studies; the SureSelect system is ideal for resolving significant questions in these disciplines.

    Clients can do their own custom design using E-array, a web-based tool that has been expanded to SureSelect. The system takes advantage of barcoding in which identifying DNA bases are inserted into the gene fragments. This permits highly targeted sequencing on a statistically relevant number of samples.

    According to Dr. Ernani, SureSelect was originally designed to work with the Illumina Genome Analyzer end-sequencing protocol but its functions have been expanded for compatibility with the Illumina Genome Analyzer paired-end sequencing and the Applied Biosystems™ SOLiD System.

  • Translational Medicine

    Click Image To Enlarge +
    The RDT 1000 from RainDance Technologies

    RainDance Technologies takes advantage of microdroplet-based solutions to empower its targeted-sequencing technology for the discovery of rare variants, according to Jeremy Lambert, product manager. “Our RainStorm™ technology produces millions of  picoliter-volume droplets per hour in a PCR format,” he explains. Each droplet behaves as a test tube that can contain a single molecule, reaction, or cell.

    “Targeted resequencing provides the opportunity to assay both common and rare variants,” says Lambert. “There is increasing interest by translational medicine researchers in understanding their contribution in complex diseases.”

    The company has designed an approach to targeted sequencing that relies on microdroplet technology. Briefly, regions of interest in the genome are identified, and forward and reverse primers for these many DNA segments are synthesized. The aqueous PCR primers for each gene segment of interest are then encapsulated in an inert carrier oil with a co-polymer surfactant, so each droplet behaves as a tiny test tube. Then genomic DNA fragments from the target are prepared, mixed together with the PCR reactants, and formed into their own microdroplets.

    The primer droplet is merged with the genomic DNA droplet, one for one. This is the equivalent of 1.5 million individual PCR reaction tubes running in parallel in a thermocycler all pooled in a single 0.2 mL reaction tube. The amplicons are then released, and the individual reactions purified and sequenced. Thus a massively high-throughput performance is achieved. 

    Raindance’s first commercial application for the RDT 1000 instrument platform is sequence enrichment, attacking relevant target-specific regions on which researchers want to focus for resequencing, for example as a follow-up to genome-wide association studies. The company’s depth of coverage provides complete resolution and supports all sequencing platforms. “We will shortly announce extensions of our platform into DNA methylation and deep-resequencing applications, which we believe will be of particular interest to researchers studying cancer and immunology,” says Lambert.

    Late last year, RainDance Technologies and CLC bio inked a partnership to develop specialized software for the analysis of targeted-resequencing data generated by workflows incorporating RainDance’s Sequence Enrichment Solution and next-generation DNA sequencing platforms. RainDance believes that by partnering with CLC bio, it can supply researchers with a solution that leverages the power of its microdroplet-based technology to provide the highest-quality results in a simple yet powerful workflow.

    As a first step, CLC bio will release an expansion of its CLC Genomics Workbench, enabling scientists to analyze data faster and more effectively when using the RainDance solution for large-scale targeted resequencing studies.

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