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Short Reads From Last Week’s “Exploring Next Generation Sequencing: Applications and Case Studies” Conference in Providence, RI

VisiGen Measures 150 Polymerase-Nucleotide Interactions in Real Time
Susan Hardin, CEO of Houston-based VisiGen Biotechnologies, gave an update on her company’s efforts to develop a high-throughput real-time single-molecule sequencing platform.
VisiGen plans to measure interactions between an immobilized DNA polymerase carrying a donor fluorophore and nucleotide triphosphates labeled at their gamma-phosphate positions with acceptor fluorophores. The measurements, which take place as the polymerase synthesizes DNA, rely on Förster resonance energy transfer, or FRET (see In Sequence 5/8/2007).
Recently, VisiGen researchers have been able to measure 150 interactions between a donor polymerase and a single type of acceptor nucleotide in real time, Hardin reported at the CHI conference.
Based on these results, the company now projects read lengths of at least 1,000 bases. The aim is to build a machine with a throughput of a million bases per second, or 100 gigabases per day.
VisiGen plans to offer sequencing services by the end of 2009, followed by the commercial launch of an instrument. Hardin told In Sequence that VisiGen expects the instrument to cost less than $500,000.
VisiGen is aiming to sequence a genome for $1,000 at about 10X coverage in less than a day, which includes reagent costs and instrument depreciation.

RainDance Multiplexes PCR for Next-Gen Sequencing
RainDance Technologies has been working on a range of applications for its droplet-based microfluidic platform, including several for preparing DNA samples for sequencing.
The company encapsulates reagents into micron-sized droplets and moves them through microfluidic channels on so-called NanoReactor chips that are preconfigured for different tasks (see In Sequence 7/17/2007).
One application is multiplex PCR, “a powerful method for genome partitioning,” according to John Leamon, a company project leader, who gave a talk at the CHI conference. In that application, droplets containing target DNA and PCR reagents are individually merged with droplets containing a primer pair from a primer library, and the PCR reaction proceeds on-chip.
Leamon showed that the company has successfully amplified nine non-overlapping adenovirus genome regions using a multiplexed primer library and is currently working on increasing the number of parallel targets to 96.
The PCR products can feed into different next-generation DNA-sequencing platforms, including those made by 454, Illumina, and Applied Biosystems. The system is “absolutely agnostic” to the downstream sequencing platform, Leamon said, but he told In Sequence that for 454’s Genome Sequencer and ABI’s SOLiD, no additional emulsion PCR step is required, simplifying the sample prep.
Another application Leamon presented is isothermal DNA amplification, for example to amplify plasmids for sequencing. He said the system has achieved yields of 300 picograms of DNA from a 6-hour incubation.
But the platform can also be used for tasks unrelated to DNA sample prep, such as qPCR, RT-PCR, SNP detection, cell sorting, or protein-based applications.
RainDance is planning to place four beta units of its system early next year. The company is also currently taking orders for commercial units, which it plans to ship in the second half of next year. The instrument will cost between $150,000 and $300,000, depending on the number of lasers and pumps.

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