Targeted Sequencing in Maize Genome Utilizing a Novel Two-Stage Sequence Capture Method with NimbleGen Arrays and 454 Sequencing Systems

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Researchers from Iowa State University, University of Minnesota, University of Florida and Roche NimbleGen (SIX: RO, ROG; OTCQX: RHHBY) have published a novel method to perform targeted sequencing in the highly complex maize genome. The method, which can be easily adapted to other genomes including partially sequenced genomes, has the potential to revolutionize genetic studies in agriculturally important species with a goal of leading to more robust and safer crops.

Sequence capture technology, pioneered by Roche NimbleGen and its collaborators, has been widely adopted in human disease studies. Historically, a required component of the capture process is blocking DNA, which prevents the repeats in the genome from inhibiting the capture process. Blocking DNA is a speciesspecific reagent that can be technically difficult to produce for each species in large quantities at highquality. To overcome this challenge, scientists developed a blockerfree, twostage protocol where the first capture stage uses an array to deplete repetitive sequences while the second capture enriches for the specific target regions. The scientists were able to demonstrate several thousandfold enrichment and high coverage of target regions in the maize genome with this twostage protocol.

Due to the large, highly repetitive nature of the genomes of many agriculturally important crops, developing a technology for unraveling them and capturing only the portions of interest is of critical importance. This study shows the innovation and power of a combined solution using NimbleGen Sequence Capture arrays and the long read sequencing capabilities of the Genome Sequencer FLX System from 454 Life Sciences, a Roche Company. The long 400 base sequence reads allowed researchers to document both single base genetic variations and longer novel sequences that are not within the captured regions in the reference genome. This ability to capture unknown sequence, by association with its known neighboring sequence, is of critical importance in a genome like maize where ~15% of the genome displays extreme sequence variation.

"The availability of a flexible sequence capture platform for plant species will provide opportunities to address many biological questions, stated Nathan Springer, Associate Professor of Plant Biology at the University of Minnesota. "We can foresee resequencing of captured gene sets that are targeted towards discovery of allelic variation that may affect important agricultural traits. The ability to document novel allelic variation beyond SNPs will be critical in plant species with dynamic genomes.

According to Dr. Patrick Schnable, Baker Professor of Agronomy at Iowa State University, "Substantial public investments in crop genomics have simplified the process of identifying chromosomal regions and genes that regulate agriculturally important traits. NimbleGen Sequence Capture allows crop scientists to easily identify valuable natural genetic variation for these traitdetermining loci ("allele mining"). As such, this technology is expected to speed the development of crops that will better withstand the environmental stresses associated with global climate change and that have other desirable characteristics."

"Including its obvious uses in agricultural systems, this technology has immediate utility in other nonfood crops like forest trees, biofuel grasses like sugarcane and miscanthus, and several nonmodel botanicals that are positioned to address interesting questions in genome architecture and gene/genome evolution. Many of these genomes are extremely large even when compared to maize, and the majority of the extra sequence is composed of repetitive DNA. Having the ability to perform targeted next generation sequencing of gene families, groups of genes, or defined regions of a reference genome, will enable discovery of the variation in genes underlying traits that are important for the forest and biofuel industries," said Brad Barbazuk, Assistant Professor of Biology at the University of Florida.

For more information on NimbleGen Sequence Capture arrays, visit For more information on 454 Sequencing Systems, visit
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