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RainDance Developing Ultra-Deep Resequencing Application for Cancer Research

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By Julia Karow

This article has been updated from a version posted June 22 to reflect several technical corrections.

A year after commercially launching its microdroplet-PCR Rainstorm technology for multiplexed sequence enrichment, RainDance Technologies is developing an ultra-deep resequencing application that will allow users to analyze gene mutations present at a frequency of 1 percent or lower within a sample.

According to the company, the application, which is currently in early-access testing and will be compatible with several next-gen sequencing platforms, will be especially useful for analyzing rare mutations in panels of genes in heterogeneous tumors, including formalin-fixed paraffin-embedded tissue samples.

It could help RainDance expand its technology's reach into cancer centers and translational research centers. To date, the firm has a couple of dozen customers, including major genome centers, medical genetic testing labs, sequence service providers, and pharma companies, such as Pfizer.

Ultra-deep sequencing — with up to 30,000- to 50,000-fold coverage — will allow users "to get to really deep regions of the cancer genome that you cannot detect, traditionally, with next-gen sequencing," said Roopom Banerjee, RainDance's president and CEO, adding that it will be complementary to whole-genome cancer sequencing efforts.

While the commercial launch of the new application is planned for the end of this year or early 2011, a first version, which will allow users to analyze mutations with a minor allele frequency as low as 2.5 percent and study pooled samples, is scheduled for launch in the third quarter.

RainDance is not the only firm offering multiplexed PCR enrichment of genomic targets — Fluidigm's Access Array IFC microfluidic chip, for example, allows users to PCR-amplify up to 48 regions in up to 48 samples. But RainDance offers a greater number of PCR reactions per sample — up to 2 million reactions, covering 20,000 regions — at a throughput of eight samples per day and chip.

Several technical differences distinguish the ultra-deep resequencing application from RainDance's currently available targeted resequencing product. For a start, it uses primer pairs with unique ligation adapters at the end that make them suitable for PCR of short DNA fragments, which often occur in FFPE samples.

Also, by running 2 million PCR reactions per sample, the sensitivity is increased. Finally, there is "some bioinformatics on the back end that is unique to cancer samples," according to Banerjee.

While customers can choose which genes they want to include in their cancer panel, RainDance has developed a primer library to amplify a panel of 142 genes that cover most major cancer pathways, which it has been providing to customers for test runs. Customers will likely include most of those genes in their custom panels, Banerjee said.

Like the existing application, the 2.5-percent-frequency product will cost on the order of $350 to $500 in consumables per sample. Pricing for the 1-percent-frequency product has not been finalized yet but will likely be on the order of $500 to $1,000. Customers also have to purchase primer libraries, which currently cost $12 per primer pair.

The RDT 1000 instrument has a list price of $225,000, but several providers now offer RainDance amplification services, including Expression Analysis, Ambry Genetics, and NCGR in the US, and Atlas Biolabs in Germany.

After a customer receives a primer library — with each primer pair contained in a droplet — genomic DNA, primers, and reagents are combined by merging droplets on RainDance's RDT 1000 instrument, followed by PCR amplification. The company supports libraries with up to 20,000 primer pairs and amplicon sizes between 150 and 600 base pairs, according to its website. A typical microdroplet PCR reaction generates at least 250 nanograms of PCR product. RainDance recommends ligating the amplicons into concatamers and then shearing these to fragments suitable in size for constructing sequencing libraries.

Currently, the chemistry for the ultra-deep sequencing application has been tailored to Illumina's HiSeq 2000 platform, Banerjee said, but RainDance is "certainly not closed to conversations with other companies."

Earlier this year, the firm struck an agreement with Life Technologies to co-market its enrichment technology for sequencing on the SOLiD system. RainDance is also collaborating with Pacific Biosciences under a partner program announced by PacBio earlier this year. And before the end of the year, there will likely be additional partnerships with other next-gen sequencing vendors, according to Banerjee.

One of several current early-access customers, and a co-developer of the application, is the Moores Cancer Center at the University of California, San Diego. Olivier Harismendy, a project scientist at the center, said he wants to use RainDance's ultra-deep sequencing to study the most commonly mutated cancer genes, with the goal to improve cancer diagnostics and treatment. "The fact is that even though many cancer mutations are known, no one has looked at them at the sub-tumor level, and how they associate with disease progression or drug resistance," he told In Sequence.

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