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Team Marries DNA Amplification and Next-Gen Sequencers to Enable Targeted Resequencing

Researchers at Stanford University’s Genome Technology Center and their colleagues have coupled a multiplexed DNA-amplification method with next-generation sequencing to resequence multiple human cancer genes in parallel and at lower cost than traditional methods.
Unlike traditional PCR, results provided by the DNA-amplification technology, also known as the selector technology, allows researchers to amplify many different DNA regions in a single reaction tube.
The researchers used 454 Life Sciences’ sequencing technology in their proof-of-concept study, which was published in last week’s Proceedings of the National Academy of Sciences. They are now testing the technology in a larger project that will use other new sequencing platforms, including Illumina’s Genetic Analyzer.
Combining multiplexed DNA amplification and next-gen sequencing could significantly lower the cost and time of large-scale, targeted resequencing projects, and make such studies possible in smaller laboratories that do not have access to the same infrastructure as large genome centers, according to the researchers.
“This is a really great (and important) step forward in the right direction,” Jay Shendure, a researcher in George Church’s group at Harvard University, wrote to In Sequence in an e-mail message last week. Shendure, who has been working on a similar amplification method and was not involved in this study, called the results “impressive.”
Methods to amplify subsets of the human genome will be crucial for targeted resequencing studies, which Shendure and others believe will initially dominate human genome resequencing.
“Even with new [sequencing] technologies, sequencing of a complete human genome will continue to be prohibitively expensive for most investigators, and most of the immediate demand will lie in targeted resequencing,” he said.
Indeed, earlier this month, Helicos BioSciences said in a filing with the US Securities and Exchange Commission that targeted genome sequencing is one of the two first applications it is targeting for its upcoming sequencing system (see In Sequence 5/15/07) .
In addition, two recent large-scale cancer resequencing studies targeted 13,000 genes in 22 samples and about 520 genes in 210 samples, respectively (see In Sequence 3/13/07)
Using Selector
For the pilot study published in PNAS, the researchers, led by Ron Davis and colleagues at the Stanford Genome Technology Center, used the selector technology to amplify 10 cancer genes comprising 177 exons from genomic DNA derived from cancer cell lines.
The technology was originally developed by scientists at Uppsala University in Sweden and is commercialized by Olink, an Uppsala-based startup company.
In traditional PCR, no more than 10 reactions can usually be combined in a single tube without getting too many undesired byproducts. Therefore, it is necessary to perform many separate reactions. By contrast, “hundreds of individual selector constructs readily can be multiplexed in a single reaction volume,” the scientists write in their article.
Using 454’s Genome Sequencer 20, they sequenced the amplified DNA and identified a number of mutations and polymorphisms, some of which were previously unknown. The researchers noted the large number of insertions or deletions they found in homopolymer regions, most of which were probably due to sequencing errors. These errors could be avoided by using a different sequencing platform, or by refining analysis algorithms and parameters, they write.
The scientists are now also improving the sequence coverage, which reached 93 percent on average per sample in their study. “We are working on various optimization schemes to be able to increase the total coverage,” said Fredrik Dahl, lead author of the study and a researcher at the Stanford Genome Technology Center, who helped invent the selector technology as a graduate student at Uppsala University. One solution is to design more selectors for target regions that prove difficult.
“But the more difficult problem with the selector technology is that some targets are more frequently sequenced than others, [because they] are more efficiently amplified than others,” he said. To fix this, he and his colleagues have developed new ligation protocols and “have some preliminary data that looks much more even in terms of amplification efficiency,” he said.
The researchers claim that their approach, which does not require the automation and personnel needed for traditional PCR coupled with Sanger sequencing, will lower the cost of targeted resequencing, but they did not provide specifics.
“In the long run, we anticipate that this is going to be a very cost-effective way of doing it, but we cannot give you a specific number now because it’s just too difficult to estimate those infrastructure costs, which are significant,” said Hanlee Ji, who leads the clinical cancer genomics group at the Stanford Genome Technology Center.
According to Harvard’s Shendure, “the biggest challenge is going to be scaling up from the exons of 10 genes to targeting the exons of all 25,000 genes.”

“Even with new [sequencing] technologies, sequencing of a complete human genome will continue to be prohibitively expensive for most investigators, and most of the immediate demand will lie in targeted resequencing.”

To make a project like this more cost-effective, Shendure and his colleagues are using oligonucleotide pools synthesized on microarrays for their amplification method, which is cheaper. By comparison, the Stanford researchers have been using the more expensive conventionally synthesized oligos because their quality is better, according to Dahl.
Shendure said Church’s group has developed a similar amplification method, which reaches a similar level of multiplexing, but he and his colleagues have not integrated it with sequencing and SNP discovery to a similar extent yet. He said he hopes their method “will have greater flexibility in terms of what is targeted” than the method used by the Stanford group.
The Stanford researchers are currently scaling up their project to 100 genes, and plan to use other next-generation sequencing platforms, as well as Affymetrix’s resequencing arrays.
“We know that this [amplification technology] will work with any of the parallel sequencing technologies, and is applicable to the system being developed by ABI, Roche, [and] Illumina,” said Ji.
Ji, who is also a medical oncologist at Stanford University School of Medicine, plans to purchase an Illlumina sequencer for his group soon and will test this platform next. “We feel that the sequencing depth off of that instrument would be ideal for what we are planning on doing,” he said.
His group would also like to test other next-generation platforms as they become available, including ABI’s SOLiD system and Helicos’ HeliScope. “We are starting an initial collaboration with one of these but we cannot be more specific,” Ji said.
“The idea is to be able to learn about these sequencing technologies comprehensively, and think about how to adapt specific, particularly clinically relevant aspects to optimize the strength of each of these sequencing technologies,” he said.
Once they have shown that they can identify somatic mutations with high confidence using the new technologies, they plan to embark on four clinical pilot projects, involving four different types of cancer. The aim is not only to find novel cancer mutations but also to develop clinically useful prognostic tests based on these mutations.
Cost will be an important factor in deciding which new technology to use in these clinical projects. “To be able to integrate these technologies into clinical studies, the cost has to reach some reasonable level,” Ji said. “You can’t run a clinical sample and charge $5,000 to sequence 100 genes; that’s just not practical,” he added. “So driving the cost down is really key towards widespread adoption of any of these sequencing technologies, whether they will be arrays or parallel sequencing.”