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GigaGen Developing Droplet PCR System for Single-Cell Sequencing, Will Target Immune Applications

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By Molika Ashford

Still early in its efforts, GigaGen this week discussed its plans to develop and commercialize a microfluidic platform for single-cell analysis, which the company hopes to market as a tool for sequencing immune cells to monitor and diagnose disease.

After fluidics company Dolomite announced last week that it plans later this year to begin selling a droplet-merging chip developed as part of GigaGen's larger droplet microfluidics system, GigaGen CEO David Johnson told PCR Insider that the company recently closed a small seed financing round with Sequoia Capital and Claremont Creek Ventures, which it plans to use to support development and commercialization of the full system. He declined to disclose how much money GigaGen raised.

Johnson described the company's goal for the system, called "Cell-Seq," as "massively parallel single-cell sequencing." He said the system will be able to analyze millions of individual cells using a special multiplex PCR coupled with standard next-gen sequencing technology.

"By performing multiplexed PCR on single cells encapsulated inside of droplets, we are able to measure expression of dozens of loci for each single cell," Johnson said.

Though the technology shares some basic design premises with other droplet PCR technologies like those of RainDance and QuantaLife, and shares some of its market targets with other immune sequencing efforts, Johnson said GigaGen believes its Cell-Seq will fill a unique niche.

"The basic premise [is] a droplet technology, which is a lot like RainDance or QuantaLife in terms of the premise [of the technology], but we are doing things very differently because we are not interested in what those guys are interested in, [which is] digital PCR or sequence capture," he explained.

"[Ours is] a much more complicated problem because just putting clean DNA or RNA … in droplets, that's really not that hard, but a cell is a mixture of lipids, proteins, DNA, RNA, polysaccharides, and so you have to do a lot of complicated stuff in addition to just lysing the cell in the first place," Johnson said.

"You can't just throw a cell in with polymerase and expect to get amplification. There are more complicated steps that have to happen," he added. For example, "RainDance only measures a single locus in each droplet … and does not use cells as input, [so] our technologies' input and output are completely different."

GigaGen’s system, Johnson explained, involves lysing cells inside individual droplets, adding a proprietary PCR mix, and doing "advanced multiplex PCR."

"This enables us to later take the PCR products out of the droplets and do next-gen sequencing of the product," he said.

According to Johnson, any next-gen sequencing platform could be compatible with GigaGen's technology. The company is currently working with Illumina machines, but "there is really no reason we couldn't use any platform," he said.

Immune sequencing is already the target of other companies, most notably Sequenta, Johnson said. However, he said, GigaGen believes its single-cell droplet approach should offer advantages over such approaches. "By analyzing single cells we can add a layer of functional profiling. Our technology is like combining flow cytometry" and the kind of cell sequencing performed by companies like Adaptive Biotechnologies and subsidiary ImmunoSeq into a single assay, he said.

Johnson explained that single-cell analysis using GigaGen’s droplet multiplex PCR system could fill a niche that is currently served only by flow cytometry but with the addition of next-gen sequencing's deeper analysis.

"There are a lot of problems with flow cytometry in terms of multiplexing, and in getting any type of sequence data. You can't get sequence data from staining with an antibody," he said.

For example, if researchers want to explore the clonal expansion of different types of immune cells and to know the fractions of different clones, "no technology can do this right now," he said.

Johnson noted that GigaGen's platform will enable "physical linkage of those expression loci that tell you whether you are a [regulatory T cell] or a T-helper cell — linkage between the T cell receptor and those other loci… such that you can then put the library on the next-gen sequencing system and sequence through the T cell receptor all the way to the particular expression locus you care about."

“Every T cell has a particular genetic sequence in its T cell receptor. Every B cell has a particular sequence in its immunoglobulin," Johnson added. "You don't get those data with flow cytometry. You’re missing a lot of information … And if you are looking to develop high-value diagnostics, looking at those particular clones gives you more specificity, more personalized diagnosis for the individual."

GigaGen is not the only company working on single-cell genomics. Fluidigm reported this month that single-cell applications are currently driving a large proportion of sales of its BioMark HD system (PCR Insider 2/16/2012).

Johnson said that while this is a great system, droplet-based analysis goes further to meet the needs of research areas that require the ability to analyze millions, rather than hundreds of single cells.

In order to gain a full portrait of a system as complex as the immune system, "immunologist end-users demand to look at millions of cells." Therefore, technologies that measure 100s or 1000s of cells "fall far short," he said.

Another application the company is working toward in the immune cell space, Johnson said, is minimal residual disease monitoring.

He said the company's aim there is similar to other immune sequencing efforts like Sequenta's, but GigaGen hopes its system will offer more specificity in being able to not only track leukemic clonal cells, but also offer functional information through its single-cell approach.

Additionally, GigaGen believes the system could be a good tool for screening circulating tumor cells.

Johnson said CTCs "can be as rare as one in 1 billion blood cells, and methods that use antibodies to sort cells and then analyze them genetically are slow, cumbersome, and inaccurate. Measuring 100s or 1000s of cells again doesn't cut it." He claimed GigaGen's system can "screen through millions of cells, without using antibodies."

According to Johnson, GigaGen is working with two academic groups — one at City of Hope, and the other at Stanford — which are currently using the company's technology.

He declined to detail the research groups involved, but said the Stanford team is working on a project to examine the immune system in patients who have undergone transplantation.

While the company's recent seed funding was small, Johnson said GigaGen is hoping to raise a larger series A financing round sometime this summer.


Have topics you'd like to see covered in PCR Insider? Contact the editor at mashford [at] genomeweb [.] com.

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