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Cracking the Code of Mendelian Disease and Cancer

Wed, 09/15/2010 - 5:43am
Fred P. Ernani, PhD; SureSelect Platform Business Manager; Agilent Technologies,Santa Clara, Calif.
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The advent of high-throughput DNA sequencing and the completion of the Human Genome Project was supposed to usher in the era of personalized medicine, where the treatment of disease could be tailored toward one’s genetic predisposition and predicted response to drug therapies. However, this goal has still not been realized. While massively parallel, or “next-generation,” sequencing technologies have increased the scale of sequencing by orders of magnitude, additional sample preparation techniques are necessary to make the most of current sequencing methods. The Agilent SureSelect platform addresses this need through a hybrid target selection method developed in collaboration with researchers at the Broad Institute.1

The SureSelect platform uses a well-established hybridization method Agilent Technologies whereby ultra-long RNA oligonucleotides, or “baits,” are used to fish targets of particular interest out of the genome for DNA sequencing. Baits are designed to be complementary in sequence to the genomic targets. When the baits and targets are combined in a single tube or microtiter plate well, they form a strong RNA:DNA hybrid molecule. The RNA baits are biotinylated at several places throughout the molecule so that the hybrids can be efficiently pulled down by streptavidin-labeled magnetic beads and separated from other genomic fragments that are not of interest. The NGS instrument is then loaded with targeted regions of the genome, ensuring that only genes of interest are sequenced, saving significant time and money. Targeted re-sequencing using the SureSelect capture kit can generate the high level of sequence coverage necessary for mutation discovery, validation, and profiling. Researchers can design their own kits targeting from below 200 Kb to up to 7 Mb and beyond with Agilent’s eArray, a free design tool, or select from a variety of pre-defined kits such as ones that target the entire human exome. By combining these products with multiplexing support, scientists can realize the full power of next-gen sequencing and perform studies that were previously too costly. 

The SureSelect platform has already allowed researchers to identify several mutations responsible for Mendelian disorders, as well as inheritable forms of cancer. Since there is a substantial body of evidence linking mutations in kinases to the development of cancer, 2-6 Agilent entered into a collaboration with Dr. René Bernards at NKI in the Netherlands, to develop the SureSelect Human Kinome Kit. This kit, released in September of 2010, will enable cancer researchers to study mutations in greater than 500 kinase genes as well as several other cancer-related genes. Such studies may lead to more efficient development of cancer therapies. Since this kit supports multiplexed sequencing, researchers will be able to fully exploit the increasing sequencing capacity of their instruments.

About the Author
Dr. Ernani studied microbiology and immunology at the University of Texas Health Science Center at San Antonio.  He has worked for more than 10 years for companies developing and marketing tools to enable geneticists to efficiently sequence DNA.
 
References
1. Gnirke A, et al. Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing. Nat Biotechnol. 2009;27(2):182-9. Epub 2009 Feb 1.
2. Pierce S, et al. Mutations in the DBP-Deficiency Protein HSD17B4 Cause Ovarian Dysgenesis, Hearing Loss, and Ataxia of Perrault Syndrome. Am J Hum Genet. 2010. Epub ahead of print.
3. Lalonde E, et al. Unexpected allelic heterogeneity and spectrum of mutations in Fowler syndrome revealed by next-generation exome sequencing. Hum Mutat. 2010;8:918-23.
4. Sun Y, et al. Terminal osseous dysplasia is caused by a single recurrent mutation in the FLNA gene. Am J Hum Genet. 2010;87(1):146-53.
5. Walsh T, et al. Detection of inherited mutations for breast and ovarian cancer using genomic capture and massively parallel sequencing. Proc Natl Acad Sci U S A. 2010;107(28):12629-33. Epub June 28, 2010.
6. Hoischen A, et al. De novo mutations of SETBP1 cause Schinzel-Giedion syndrome. Nat Genet. 2010;42(6):483-5. Epub May 2, 2010.

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