Right on target

MADISON, Wis.—Using technologies that include targetedenrichment with Roche NimbleGen's Sequence Capture and ultra-deep sequencingwith the Genome Sequencer FLX System from 454 Life Sciences (also a Rochecompany), Roche Applied Science is providing what is described as "a completesolution for targeted resequencing of the human genome to further ourunderstanding of disease associated regions."

Roche and its partner, National Cancer Institute (NCI)contractor SAIC-Frederick Inc., will assess the ability of these technologiesto sequence the genomes of cancer research samples. As one example, in ajust-published collaborative study between the two partners and the NCI, adetailed map of common genetic variation in the genomic region surrounding theKLK3 gene was generated, which should be useful for fine-mapping theassociation signal as well as determining the contribution of this locus toprostate cancer risk and/or regulation of PSA expression, the authors state.

Together, the NimbleGen Sequence Capture arrays and 454Sequencing system offer a complete solution by providing comprehensive,relatively unbiased coverage across the desired target enrichment region, saysTom Jarvie, technical applications manager at 454 Life Sciences. Furthermore, henotes, the system is not limited to SNPs, but covers the full range of genomicvariation. Known disease-associated regions can be quickly converted intocustom-designed target enrichments.

"Our ability to understand the impact of particular regionsof the human genome with cancer phenotypes is rapidly advancing with the adventof next-generation sequencing technologies," says Joseph Boland, dedicatedscientific operations manager of the Core Genotyping Facility atSAIC-Frederick. "Our intent is to implement Roche NimbleGen's solution-basedsequence capture technology, SeqCap EZ, to rapidly advance our next-generationsequencing projects and, in turn, increase our knowledge of cancer genetics."

The long, accurate reads of the ultra high-throughput GenomeSequencer FLX system provide a unique opportunity to identify classes ofvariation that are not possible with short read sequencing technologies, TomJarvie says. The long reads enable the identification of insertions anddeletions, the ability to provide haplotype information on two or morevariations (to determine if the variations are linked on the same chromosome),and the ability to discern between variation within nearly identical sequences.The nearly identical sequences are present in closely related genes (genefamilies, paralogous genes) and within genes that have closely relatedpseudogenes elsewhere in the genome. 

Additionally, all target enrichment technologies must omitareas, such as repeats, from the pool of capture oligos—even when these regionsare within the desired target enrichment region. However, these omitted areasare still important to the overall experimental success. NimbleGen SequenceCapture technology successfully captures fragments that border the omittedareas and the long, 454 Sequencing reads span the gap thereby providingcomprehensive coverage of the entire target region, Jarvie notes.

"The target enrichment, coupled with ultra high-throughput,long-read sequencing, allows researchers to focus in on the regions of interestin the genome that are either biologically significant, such as the wholeexome, or a small region that is known from other studies to be of interest fora particular type of cancer. By focusing the sequencing in this way, the datasets are much smaller, quicker to generate, and more readily and quicklyinterpreted than whole genome data sets," Jarvie states.

Kary Staples, manger of global marketing communications atRoche NimbleGen, notes that the SeqCap EZ system was developed by RocheNimbleGen within the past year and is targeted for mass-market launch at theend of October. Some of the differentiators of this product are, it offers morecapture oligos (up to 2.1 million) than other technologies, and this togetherwith an optimized design algorithm (or empirically optimized and validatedprobes), increases the capture uniformity. Compared with other technologies, itachieves better coverage with less sequencing due to the large number of probesand the enhanced uniformity of the capture. In addition, the SeqCap EZ workflowis easily implemented and scalable to a 96-well microplate to achieve 96discrete captures.