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Sandia Builds Automated Microfluidic NGS Library Prep System; Modules Provide Flexibility

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Scientists and engineers at Sandia National Laboratories have built a prototype of a modular microfluidic library preparation platform that will allow users to process small numbers of DNA samples for next-gen sequencing in an automated fashion.

The device, which can be easily reconfigured for different applications, only requires nanogram amounts of DNA. While it currently produces libraries for Illumina sequencing, the system could be reconfigured for other types of sequencing platforms.

At the beginning of the three-year project under which the prototype was developed, robotic liquid handling systems to automate NGS library prep for thousands of DNA samples already existed, but "there was nothing automated for doing tens of samples," said Ken Patel, manager of advanced systems engineering and deployment at Sandia.

The goal of the project, called RapTOR, for Rapid Threat Organism Recognition, was to develop a new approach for identifying unknown pathogens (PCR Insider 5/12/2011). "The idea is that you could take samples from patients who are sick that are exhibiting certain signs, and then when traditional tests like PCR fail, you use sequencing as a secondary measure to quickly discover what that disease is," Patel said, "and to do that in a very automated, fast way, so it could be useful for making decisions on what to do regarding this outbreak or potential epidemic."

"We set out to develop a platform, based on microfluidics, that is good for processing small numbers of samples," he told In Sequence. "The sweet spot is medium to low throughput where automation is welcome, but there is such diversity between the samples that it would be hard for you to have just one cookie-cutter-type system that can do everything."

Patel and his colleagues recently published a description of the prototype in PLoS One, along with examples of its use to prepare NGS libraries of E. coli, Klebsiella pneumoniae, and human DNA.

The device consists of a central droplet-based digital microfluidic hub for routing and mixing liquids, and several peripheral modules for specific steps of the workflow — such as enzymatic reactions, sample clean-up, PCR, and size selection — that are connected to the hub through a capillary interface.

"The unique thing about our platform compared to, say, some of the traditional approaches in microfluidics is the hub design," Patel explained. "Rather than trying to integrate everything onto a single chip or a monolithic device, we are taking a different strategy where we are just focusing on integrating multiple components together through this droplet interface."

The digital microfluidics hub functions like a robotic pipette that transfers liquids between the modules and integrates their functionalities, "so we leave these modules to do what they are really good at," he said.

One advantage of using microliter-sized droplets, rather than the nanoliter volumes used in traditional channel-based microfluidics, is that many existing protocols scale well to the microliter volume without the need for extensive modifications, Patel said. His team found that a lot of potential users "have a protocol that they're very happy with, they just want to automate it, they don't want you to reinvent the assay," which their system allows them to do.

And while some of the modules, for example the thermocycler, are "quite unique and different" from traditional instruments, they use the same principles as existing assays. "We're not inventing something new," Patel explained. "It's very similar to what you would do traditionally."

To move droplets around, the hub currently uses the same electrowetting technology that has been developed and commercialized by Advanced Liquid Logic, a company that Illumina recently acquired to use the technology to develop its own NSG sample prep devices (IS 8/13/2013). Patel said the Sandia system could in principle use different technologies for transporting droplets, but he declined to provide more details at this time.

NuGen Technologies last year launched an automated NGS library prep system called Mondrian SP+ that uses ALL's digital microfluidics technology and was developed in collaboration with the company (IS 4/17/2012).

Unlike ALL, which converts many steps of the sample prep process to the droplet format, Sandia's platform only performs parts of the workflow with droplets. "What we're rather doing is using the droplet for what it's really good at, moving components around, orchestrating, navigating, basically connecting everything together," Patel said. "Things that are very hard to do, like PCR, magnetic bead extraction, purification, electrophoretic separation, which you can't do in a droplet anyway, we do off the device, connected through the capillary interface." This, he said, makes the platform very flexible, because individual modules can easily be added or replaced.

"Our technology augments [ALL's] work very well," he said, and Sandia has talked to ALL, as well as others, about potential licensing and commercialization of its technology, though nothing has been finalized yet.

In addition, the researchers have worked with Eureka Genomics, which is developing NGS-based assays, "to work out a strategy to incorporate some of our related Sandia technology into their pipeline," Patel said, and they have collaborated with Joe DeRisi's group at the University of California, San Francisco on informatics and sample sharing.

Sandia's capillary interface, which allows samples to be transferred on and off the hub, is patent-protected, and the lab has applied for IP protection for aspects of its PCR technology. "We're open and ready for licensing opportunities and discussing and pushing collaborative events forward" in order to develop the system beyond the prototype stage, Patel said.

In their paper, the researchers configured their system for Illumina's Nextera sample prep protocol, which involves transposase-mediated DNA fragmentation and tagging, DNA clean-up, limited-cycle PCR, and size selection.

The bacterial libraries generated by the platform were sequenced in-house on an Illumina MiSeq, resulting in about 170x coverage depth and even coverage across the genomes. For E. coli, they achieved "high-quality" sequencing data, compared to conventional library-prep methods. They also applied their system to a novel strain of Klebsiella pneumoniae, where the resulting de novo assembly was poorer than for E. coli but still yielded some interesting insights regarding plasmid DNA, transposons, and antibiotic resistance.

Since the paper was submitted, the researchers have improved the system in several areas, including making the digital microfluidic component and fluidic interface disposable, developing a better feedback loop system, and a new approach to thermocycling that is more convenient, more thermally efficient, and easier to integrate. They have also devised different ways to analyze components on the platform in situ, for example by fluorescent detection, and to quantify the components.

Patel said his team is now working on adapting other NGS library prep protocols, such as RNA-seq, for the platform. The system would also be "very applicable" to bead-based library prep protocols for the Ion Torrent platform, which Sandia does not currently have in house.

Beyond next-gen sequencing, the device could be used for other applications, and Sandia has already explored it for analyzing live cells in droplets and RNA from blood. It has also used the system to prepare samples for other analysis platforms besides sequencing, such as mass spectrometry.

"One of the benefits of this technology is that it's very versatile," Patel said. "Our intentions are to impact sample preparation on a broad scale, for a variety of different molecular biology protocols."

In particular, the group is interested in developing a portable device that could be used outside of a laboratory in areas such as DNA forensics, an effort that Patel discussed with PCR Insider earlier this year.

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