To enable the rapid sequencing of unknown pathogens, researchers at Sandia National Laboratories are creating an automated, microfluidic-based sample preparation process for next-generation sequencing from unascertained biological samples.
The single-step sample prep system is part of Sandia's Rapid Threat Organism Recognition, or RapTOR, project, which is geared toward developing a rapid sequencing method for bioterrorism applications under a Sandia internal laboratory research directive that provides seed money for projects of interest to other government agency customers.
The sample prep component of the project consists of a microfluidic hub that connects a group of modular microscale sample processing components that normalize, ligate, separate, and otherwise prepare DNA samples from unknown pathogens.
"With [RapTOR] we want to be able to detect unknown pathogens or new, emerging pathogens — things we've never seen before — using sequencing technology," said Kamlesh Patel, one of RapTOR's team leaders.
While sequencing has become faster and easier with next-gen systems, the pre-sequencing steps can still slow down things down by days or even weeks — too long a wait where the discovery of unknown pathogens is concerned, Patel said. The goal of RapTOR is to scale down and automate the traditional steps of sample prep in order to more quickly identify novel or modified pathogens in the event of a bioterrorism attack.
"The idea is that you can sequence everything that you have, and when you find a sequence you know is real but doesn’t belong to any taxonomy, you can go in and investigate very quickly that this is a new species or that this was an E. coli that’s been modified, [and] here are some… events where someone has gone in and tinkered with the genetic code," Patel said.
"Hopefully someday … [someone] could just put in a DNA sample and press a button … and then come back when he's ready to look at the data," Patel added. "Our goal is to have a platform that we can deliver to say, the [US Centers for Disease Control and Prevention], where somebody with minimal training can operate this."
Though the RapTOR project as a whole involves several areas, including the development of protocols for sequencing and post-sequencing bioinformatics, the automated sample prep system has been a central achievement, especially the design of the digital microfluidic hub, which routes sample from one step to another in the form of droplets.
"Rather than put it all on one microfluidic chip, or one fluidic channel, we are developing this interface that takes modules and connects them together... It's a PCR machine, a normalization module, a separations module all connected together via a droplet," Patel said. A finished sample would come out of this modular system and go right into a sequencer, he explained
In addition to creating the hub, Patel said, researchers have also "proven each of the [modular] components of the full prep independently," and are working to connect them.
"We can do PCR at the micro scale [as well as] separation, cleanup, and fragmentation," he said. "So what we're working on now is [doing] all of these in concert. For the most part, we are adapting proven biological protocols at the bench top, but there are some modifications we have to do to go to the sub-microliter scale."
Normalization is a particularly important aspect of RapTOR's prep system, because DNA samples from people sickened by an unknown pathogen are predominantly host DNA, which must be suppressed for pathogen DNA to stand out, Patel said.
"When you think about a clinical sample, let's say a blood sample or a nasal swab from a clinically sick patient, 99.99 percent of the DNA you're going to sequence is really human. And maybe that .01 percent is going to be the pathogen. You may just get lucky and find it and that does happen, but it's kind of a poor use of the awesome power and the bandwidth a sequencer can provide," he said.
In the RapTOR system, two techniques are used to suppress human DNA, according to Patel: normalization with hydroxyapatite chromatography and a "bead-based capture technique where we capture the human exome and remove it from the sample."
To normalize, "basically [we] take the entire DNA population and actually melt it at 95 degrees so everything turns into single strands," Patel said. A droplet moves the sample from a heating area to another region where it is cooled to 65 degrees. As the DNA cools, "you get a partial re-hybridized population where the high-abundance components have a higher statistical chance of finding [their] complementary pair and will hybridize, and the rare fractions will not find [their complement] and will remain a single strand," Patel said.
"Once we have a partially hybridized population, we separate it using a hydroxyapatite separation column," he explained.
Using the two suppression steps in parallel creates a flexible approach that can be amended depending on how much suppression researchers want to do, which is a boon to the project, Patel said. "We don't really know how much we need to do," he said. "If we remove too much of the background we may degrade our signal. The platform allows us to tune those knobs, so to speak."
In tests with known samples, this aspect of RapTOR's prep process has been shown to can increase the proportion of pathogen DNA recovered, at least as well as benchtop enzymatic methods, Patel said.
The hub also connects with components that perform other preparation steps. Patel noted that sequencers such as the Illumina Genome Analyzer require DNA to be fragmented into lengths of about 200 to 300 base pairs. RapTOR's sample prep uses enzyme-based technology to do this fragmentation, he said.
Ligation and barcoding for multiplexing are also connected to the hub, as are cleanup steps and a PCR module.
The small scale offers benefits, Patel said, for example smaller sample sizes, lower volume amounts, and speed.
Benchtop PCR takes well over an hour, but "at the micro scale you can do PCR in less than ten or fifteen minutes," and the process requires less sample manipulation than at the macro scale, he said. "You can combine a lot of the cleanup steps together whereas on the bench top you have to do those separately."
Patel said that the team has been testing the systems by running one half of the modules and then the other. The remaining challenge is to get the whole group working together. "We haven't fully integrated the entire system, but I anticipate it [will] take up space no larger than the size of a desktop computer, at best," Patel said.
According to Patel, the group has applied for several patents on the droplet manipulation aspect of the platform and its interface, but they are still pending. He said the group is looking for commercial partners who might want to take the technology to the next step.
"We've talked to folks at Illumina and Pacific Biosystems who are interested in our general approach," he said.
In addition, the researchers have received funding from the US Army Criminal Investigations Lab to spin off the tech in another direction: genotyping instead of genome sequencing. "The idea is that we could build a portable system that could [enable] DNA fingerprinting in the field," Patel said.
Patel mentioned the recent DNA fingerprinting of Osama Bin Laden (PCR Insider 5/5/2011), saying that while this would now require sending samples to a lab, a compact automated system would mean future missions could "take a DNA sample and put it into this portable genotyping system and get the results then and there on the helicopter ride back."
"As you look into the crystal ball, look down the road, you can imagine a desktop-like sequencer in all kinds of places, like a doctor's office," Patel added. "And attached to that is an automated microfluidic version of sample prep."
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