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U of Wash Team Develops Automatable Exon-Capture and -Sequencing Method

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This article has been updated from a previous version to include additional information.

Researchers at the University of Washington have improved a multiplexed exon-capture method that is based on padlock probes, making it easier to automate and to feed its products directly into an Illumina Genome Analyzer for sequencing.

The scientists believe that the new protocol, published online earlier this month in Nature Methods, can be scaled up to the entire exome and could be useful in resequencing studies involving many samples. It is unclear, however, when or whether the protocol could be commercialized as a kit.

A year and a half ago, senior author Jay Shendure and colleagues at Harvard University published an earlier version of the method (see In Sequence 10/16/2007), which uses padlock probes — also known as molecular inversion probes — derived from Agilent custom microarrays to selectively capture and amplify tens of thousands of exons in parallel and sequence them on an Illumina GA.

Although the method proved to be highly selective for its targets, only required small amounts of DNA, and was easy to scale, at the time it suffered from "two crippling deficiencies," the authors write in their current paper: fewer than one-fifth of the 55,000 targeted exons were amplified, and those that were had been represented unevenly; and heterozygous alleles were not equally sampled.

Changes to the protocol — including increased incubation times and different reagent concentrations — improved all of these aspects, bringing the capture efficiency up to 91 percent, or to more than 50,000 of the 55,000 targets.

But the scientists also showed that they can avoid the cumbersome and difficult-to-automate steps of making a shotgun library and sequence the amplification products directly. "When you want to scale to many samples, shotgun library construction is a bottleneck," said Shendure.

Direct sequencing should thus make it possible in the future to automate the exon-capture and -sequencing process. "That was the hope going into this," Shendure said.

Also, when combined with barcoding primers introduced during the PCR, it could be used to sequence the exons of many samples at once. "We are in the process of evaluating that right now," he said.

What made direct sequencing feasible is the longer reads of the Illumina platform — the UW researchers used 76 base pairs in their study — which now covers most of the targeted exons.

"We now match the gap fill lengths of the [molecular inversion probes] to the read length from Illumina," Shendure said. "If we have a long exon, we simply use multiple MIPs."

He added that there is no reason why the method should not work on other sequencing platforms, like the Applied Biosystems SOLiD or the Roche/454 GS FLX, but his lab has not used it with these systems.

In their study, the researchers tested the protocol by targeting 13,000 exons in 16 HapMap samples. But Shendure said it could be scaled up further. "We will be trying to do the whole exome," he said, or more than 160,000 exons in total.

He and his colleagues also reduced the amount of required input DNA to 750 nanograms, though "we can probably go lower than that," Shendure said. He also pointed out that the approach might work on "somewhat" degraded DNA, saying that "we have some preliminary data that suggests that that may be the case."

In the future, the scientists want to use the new method in projects currently in the planning stage that involve large numbers of samples and well-defined targets. "That's a place where it could be particularly useful," Shendure said. Researchers at the National Human Genome Research Institute led by Eric Green and Leslie Biesecker have also been evaluating the method, he said.

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But while the exon-capture method might be useful, its path to commercialization is unclear at this point. Intellectual property covering padlock probe technology in particular might hold up the process. "The IP landscape in this area is a bit of a minefield, and I don't totally understand it," Shendure said.

In their research, the UW researchers used oligonucleotide libraries provided by Agilent Technologies under an early-access program. Agilent recently commercialized another genome capture, or enrichment, method called the SureSelect Target Enrichment system, which resulted from a similar collaboration with the Broad Institute (see In Sequence 2/24/2009).

Earlier this month, Fred Ernani, senior product manager for emerging genomics applications at Agilent, told In Sequence that while the company does not always commercialize technology resulting from collaborations involving its oligo libraries, "we are continually evaluating their potential to become products" (see In Sequence 4/14/2009).

If commercialized, exon-capture padlock probes would likely compete with existing products for exon enrichment, such as Roche/NimbleGen's recently launched Sequence Capture 2.1 Human Exome microarray.

Shendure has experience with Agilent arrays for array-based capture but said the jury is out on which one is better. "We have had great luck with both. I would not say we have a strong preference at this point," he said.

However, he said one factor for deciding which method may be more suitable is the size of the project. "A lot of it depends on how many samples you are doing: If you are doing two exomes, arrays maybe make the more sense, [but] if you are thinking about 1,000, there needs to be more thought about [how] to make it really scalable and automatable."

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