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Next-Gen Sequencers, Led by 454's GS20, Gain Ground in Small-RNA Analysis

NEW YORK (GenomeWeb News) - As new DNA-sequencing technologies enter the market, vendors and users are seeking applications other than whole-genome sequencing and even emerging ones such as mRNA-expression profiling.

They might not have to look far.
The use of sequencing to analyze small RNAs is gaining traction. "I think there is enormous potential in this area," Jim Russo, associate head of sequencing and chemical biology at the Columbia University Genome Center, told GenomeWeb News in an e-mail message last week.
Small RNAs are regulatory RNAs, generally between roughly 20 and 30 nucleotides in length, that include small interfering RNA, microRNA, and other classes. Over the last two months, at least three papers have been published in high-profile academic journals describing how researchers have used a next-gen tool to study small RNAs. Vendors without commercialized platforms are also either developing, or at least considering, small-RNA applications for their technologies.
"It seems to be a pretty hot application these days," said Bill Spencer, director of worldwide systems sales for 454 Life Sciences, whose GS20 sequencer was used in the three studies.
For example, researchers at Cold Spring Harbor Laboratory earlier this month published a paper online in Nature describing how they used the GS20 to characterize a new class of small RNAs, called piRNAs, in mouse testes. The scientists created more than 87,000 reads, around 53,000 of which they classified as candidate piRNAs.
According to Greg Hannon, whose group published the paper, several reasons "make [454's instrument] almost perfectly suited" for analyzing small-RNA sequences. For a start, he said, sample preparation is easier than producing concatamers, or linking up several templates, which researchers have been using to make small-RNA Sanger sequencing more economical. "That has proven for some people to be a tricky part of the process," Hannon said. "Doing 454 sequencing, you don't need to do that."
Secondly, the GS20 provides short reads at higher throughput than Sanger sequencing. For small-RNA sequencing, read length in the low- to mid-30 bases is sufficient, Hannon said. 454's instrument currently provides 100-bases reads.
Columbia's Russo, who provided Sanger-based sequencing for a similar study of piRNA by Thomas Tuschl at Rockefeller University, which also appears in Nature online this month, concurs that producing concatamers "can be difficult for investigators who do not do this routinely."
He pointed out, though, that 454's system is currently limited by its accuracy, which could become a problem with rare RNAs. To make up for this, he said, Hannon's group required higher throughput sequencing. However, Hannon maintained that the lower accuracy did not matter.
"We get so many reads we just throw out everything that doesn't perfectly match the genome, and we are still left with a depth that is so much greater than what one would normally get for a similar amount of money from conventional sequencing," said Hannon.
454 does not market the GS20 for small-RNA analysis but researchers use the technology for that application and the company provides small-RNA sequencing services to a number of investigators, Spencer, the sales director, said. 454 also plans to write an application note for small-RNA sample preparation, and provides a protocol developed by one of its customers to users wanting to sequence regulatory RNA.
According to Russo, next-gen sequencing companies hoping researchers will use their instruments to analyze small RNAs need to address two issues: read length and accuracy. Reads must be long enough to cover the sequence of small RNAs -- usually between 20 and 30 bases -- and accuracy needs to be good enough to locate their sequences to the genome unambiguously, and to quantify them accurately. This is especially problematic for rare RNAs, he said.
However, Russo said he is optimistic about the long-term potential of next-generation sequencing for small-RNA analysis. "In the end, if any of the methods become really cost-effective ... their potential for running tens of thousands of reactions at once and in relatively short time could well respond to the issues of even the low-abundance RNAs."
On Deck
Other next-gen sequencing companies are also embracing small-RNA sequencing as a potential application and promise lower sequencing costs and higher throughput.
"Based upon our experience with MPSS, we have been able to come up with protocols that are appropriate for studying small RNA using the SBS technology," said Gary Schroth, R&D director for gene expression at Solexa. Sample preparation, he said, will be much simpler with Solexa's new sequencing platform than with MPSS, and the entire analysis is expected to take less than a week compared to at least six weeks with the older platform.
In fact, Solexa, which is in the midst of an early access launch of its instrument, will initially sell three sample-prep kits for its instrument: one for shotgun sequencing, a second for tag-based gene-expression profiling, and a third for small-RNA analysis. "We are working on those three applications and the products to support those three applications," Schroth said. "We see a lot of interest for small RNA."
Helicos BioSciences also wants a piece of the pie and says one advantage of its system is its sample-prep method, which is "straightforward and simple" and does not involve amplification, according to John Boyce, the company's senior director of marketing. "We are interested in all applications regarding gene expression, and we are definitely interested in the regulatory effects of RNA," he said. "At this moment, we are talking with people."
According to Kevin McKernan, president of Agencourt Personal Genomics, which is soon to be acquired by Applied Biosystems, his company is also considering the small-RNA and the "more traditional" mRNA expression-profiling markets.
Weak Revenue Stream?
In general, however, small-RNA analysis might not be the most important application for the new sequencing technologies in terms of revenue potential. At a recent presentation to investors, Solexa valued at $77 million the research market for small-RNA analysis that its instrument would address. By comparison, DNA sequencing was $473 million, whole-genome gene expression was $342 million, and array validation was $135 million.
Still, small-RNA analysis is an expanding market. "You go to a meeting and you already see a lot of people talk about deep sequencing of small RNAs," CSHL's Hannon said. "I think [our Nature article] is just the first of many papers that you are going to see come out where people have used this kind of technology to analyze small RNAs from all sorts of different sources."

Julia Karow covers the next-generation genome-sequencing market for GenomeWeb News. E-mail her at [email protected].

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