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NimbleGen, Partners Develop Array-Based Alternatives to PCR for Genome Resequencing

Roche NimbleGen this week published two separate papers in collaboration with academic research teams describing how its microarray technology can improve upon PCR for sample preparation in large-scale genome resequencing projects. 
The company plans to make the methods — co-developed separately with researchers at Baylor College of Medicine and Emory University and described in two papers published in the advanced online edition of Nature Methods this week — available to all of its customers by early 2008.
The first method, called sequence capture and described here, was developed by a team led by Richard Gibbs at Baylor and enables users to select targets for high-throughput sequencing on 454 Life Sciences’ FLX instrument.
The second method, developed by Michael Zwick’s lab at Emory and described here, is called microarray-based genomic sequencing and allows scientists to extract and enrich specific large-sized DNA regions, then compare genetic variation among individuals using DNA resequencing methods.
NimbleGen expects both methods to reduce or eliminate the use of PCR in experimental workflows, and to help reposition its high-density, long-oligo arrays for more integrated projects involving next-generation sequencers.
The need to better integrate its arrays with sequencing technology has been a goal for NimbleGen, which Roche acquired in August, six months after it picked up next-gen sequencing shop 454 Life Sciences. Roche has stated that it views the two platforms as highly complementary (see BAN 6/26/2007).
Roche NimbleGen President Stan Rose told BioArray News in an e-mail this week that the sequence capture and MGS methods are still being honed for launch but both should become available to all users next year. “Although these results are very exciting and clearly demonstrate the power of this approach, the technology is still in development. Commercial products and services should be available early in 2008,” he wrote.
Rose explained that the sequence capture technology uses Roche NimbleGen’s maskless array synthesis technology to synthesize long DNA probes that are capable of targeted capture and release of genomic regions. Once a sample genome has been fragmented, the entire sample is exposed to NimbleGen’s sequence capture microarray. Genomic regions that are of no interest are then washed away, while the targeted regions are released and then subjected to processes that enable massively parallel next-generation sequencing using the 454 FLX instrument or other resequencing methods. 
According to Rose, the “ability to rapidly synthesize very large numbers of probes and to quickly modify designs to meet customers’ specific needs provides significant advantages over ... other array platforms for both development and application of sequence capture technology.”
The company expects this ability to reduce the cost and time associated with PCR in genome-based disease research.
Researchers have typically relied on PCR for selecting parts of the genome for resequencing to enrich for specific DNA fragments, but PCR is limited in the length of sequence it can amplify, and is difficult to scale or multiplex for the enrichment of thousands of fragments. Baylor’s Gibbs said in a statement that the “new technology will replace PCR for many purposes” and that “if the aim is to sequence a whole genome for everybody, this is a huge step in that direction.”
According to Rose, NimbleGen scientists are “collaborating closely” with 454 Life Sciences on sequence capture technology development and optimization for 454 sequencing. “The nature of the 454 sequencing technology is such that synergies are created between their ‘long-read’ capabilities and the conditions required for optimum enrichment performance,” Rose wrote. 
Breaking the PCR Bottleneck
Rather than being a fully separate technology from the sequence capture method, the MGS method developed by the Zwick Lab at Emory is expected to complement NimbleGen’s sequence capture arrays once they become available.

The “ability to rapidly synthesize very large numbers of probes and to quickly modify designs to meet customers’ specific needs provides significant advantages over use of other array platforms for both development and application of sequence capture technology.”

According to the Nature Methods paper, MGS uses oligos arrayed on a chip to directly capture and extract the target region from the genome. Once the target is selected, resequencing arrays or other sequencing technologies can be used to identify variations.
Zwick, a coauthor on the Nature Methods paper, told BioArray News in an e-mail this week that the MGS method could be useful to other researchers because it is more cost-effective and efficient than current methods that use PCR or bacterial artificial chromosomes.
“Existing methods would require a pair of PCR primers for each 500 bp fragment that one would want to amplify and then sequence,” he wrote. “While this method works well for a small number of fragments, when dealing with large numbers of fragments, it becomes difficult and arduous to optimize and perform routinely,” wrote Zwick. 
He added that “MGS eliminates the needs for a vast number of unique PCR primers, and can allow the isolation of large genomic regions while only requiring a single PCR reaction after the genomic selection has been completed.”
Zwick wrote that this sample preparation method could be useful for NimbleGen resequencing arrays or the 454 platform.
“It depends upon how [researchers] intend to do the resequencing,” he wrote. “We demonstrated that MGS works very well with chip resequencing [but] others have demonstrated that it works with 454. We are pursuing other sequencing platforms to make the protocol of general utility for next-generation sequencing platforms,” he wrote.
According to NimbleGen’s Rose, “MGS is currently being performed with NimbleGen arrays for both capturing targeted DNA fragments and then resequencing them.” He wrote that once sequence capture arrays become available next year, scientists will be able to use them to prepare genomic samples for resequencing using the MGS method developed by Zwick.
“Once NimbleGen launches optimized products and services for sequence capture, those products will provide a complementary front end for the MGS technique,” he wrote.

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