NEW YORK (GenomeWeb) – Sequencers, microarrays, and PCR platforms all have a part to play when it comes to analyzing microbial genomes aboard the International Space Station (ISS), according to Kasthuri Venkateswaran, a senior research scientist at the National Aeronautics and Space Administration (NASA) Jet Propulsion Laboratory.
Test cases for these technologies are under the auspices of NASA's Microbial Observatory Program, which seeks to monitor microbial activity aboard the space station, how microbial composition changes over time, and potential risks to the crew. Currently, that tracking is done using traditional cultures, Venkateswaran said, but since only one to ten percent of microorganisms can be cultured with current methods, it makes sense to evaluate the potential of sequencers and other genomic technologies to characterize the microbial environment on the space station more completely. However, no single technology will work in every situation so "you need all kinds of systems," he told GenomeWeb.
For example, PCR won't reveal exactly what microorganisms are in a sample or provide details about their molecular make-up, but the technology can be used to figure out microbial load, which is information that sequencing technologies do not provide, Venkateswaran said.
Meanwhile, microarrays are more of a detection technology, while sequencers offer a more comprehensive molecular picture of what is in a given sample, Crystal Jaing, a biologist at Lawrence Livermore National Laboratory (LLNL), noted. Jaing is also the principal investigator of Microbial Tracking-2, a new three-year $1.5 million NASA-funded effort to identify and study potential pathogens in samples from the crew and surfaces of the ISS. The study, which involves researchers from LLNL and various NASA research centers, will use LLNL's Livermore Microbial Detection Array (LLMDA), among other tools, on Earth to analyze pre-flight, in-flight, and post-flight samples from the space station and its crew.
Oxford Nanopore's MinIon sequencer has been cleared to go up to the ISS aboard a SpaceX crew resupply vehicle on June 24, Venkateswaran said — it was supposed to go up earlier this year on a resupply mission that was delayed due to a failed SpaceX CRS-7 mission launch last summer. Aboard the ISS, the MinIon will be used in experiments to test the feasibility of DNA sequencing in space. The same experiments will be simultaneously replicated on Earth.
The SpaceX 9 rocket will also tote two PCR platforms, BioFire Diagnostics' Razor Ex and Cepheid's SmartCycler, to the space station, he said. GenomeWeb reported previously that both platforms were part of a comparison between commercial PCR platforms, performed by researchers at NASA's Johnson Space Center, for environmental monitoring on space flights. Following their study, the researchers recommended BioFire's Razor Ex and another platform called iCubate, developed by a company of the same name.
However, Cepheid's system offers more capabilities than the other two platforms do and is flexible enough to support various microbial monitoring projects, which is why it was selected, Venkateswaran said. It also has plastic rather than glass sample tubes, so it poses less of a safety risk than Roche's LightCycler, which is also very efficient but uses a glass capillary tube that could break. Aboard the ISS, the SmartCycler's primary purpose will be microbial monitoring but it could also be used for gene expression analysis, among other projects that require PCR amplification. The Razor Ex will be used to test for a specific panel of bacteria, he said.
There are also ongoing efforts to develop and test custom microarrays for use on the ISS but these are still in the research and development stages, Venkateswaran said.
In the meantime, here on Earth, researchers will use Livermore's LLMDA microarray platform to find potentially harmful pathogens in the space station environment. The Microbial Tracking-2 project is a follow on from an earlier project, called Microbial Tracking-1, which was intended to provide a comprehensive picture of all the microbial species present on the ISS. Data from that study provides a baseline for the analysis that will be done for the current project.
LLMDA identifies microbes by matching them to short DNA oligonucleotide probes on a 1 inch by 3 inch glass slide. It accepts purified DNA from a wide array of samples including soil and air, as well as blood and other tissues.
The version of the chip that will be used for the space station project detects 12,609 species, including 6,906 bacteria, 4,776 viruses, 414 fungi, 143 protozoa, and 370 archaea, and it can analyze up to four different samples at a time. The chip processes samples in about a day and provides about 50- to 100-fold more coverage than traditional culture-based methods. "[It] is a really good technology [for] look[ing] at a comprehensive list of microbes and pathogens in a single sample without knowing what is actually in the sample," Jaing said. "We are looking for unique DNA signatures that correspond to the different pathogens or microorganisms, [so] the microbes don't have to be alive for us to identify them."
In total, the researchers will analyze 18 air samples, 24 surface wipes, and 264 body samples from three crew members — figures based on the number of samples that were collected for the Microbial Tracking-1 project. They will collect the first set of samples about six months before the astronauts leave for their mission in the spring of next year. During the flight, the astronauts will collect more samples a few days into their flight and another set of samples a few days before they come back to Earth. The researchers will collect the final samples a few days after the astronauts have returned.
Besides identifying the microbial species and strains present in the samples, the researchers will also sequence these microbes to identify potentially pathogenic mutations as well as antibiotic resistance patterns. Previous studies have shown that in some environments, once harmless microbes can become virulent. For example, a 2011 study by researchers at NASA's Johnson Space Center and elsewhere found that latent herpes viruses can be reactivated under the extreme conditions associated with space flight. "Can we identify those virulence genes or antibiotic resistance genes present in the sample ... that will help us understand how these microbes become more pathogenic?" Jaing said. Furthermore, "are there any countermeasures we can use, based on this information, to treat the astronauts?"
The researchers will use the LLMDA to analyze the collected samples here on Earth but Venkateswaran said the system is a candidate for possible use aboard the ISS. "I think it will be something really interesting to see if the microarray can stand ... microgravity," Jaing said. "I suspect it should be but it needs to be tested to make sure that it does work."
Before it can go up to the ISS, LLNL researchers need to build a more compact version of the LLMDA, Jaing said. Specifically, the accompanying array scanner would need to be much smaller and lighter. Also, the sample prep and other protocols would need to be much more automated and tightly integrated. "It can be done," she said. For example, they could perform both the sample prep and probe hybridization on a single chip. "That's something that I think will be great for a future generation of the array," she said. "We'll definitely consider that for future projects."
Meanwhile, there is still work to be done to prep both the nanopore sequencer and the PCR systems for space use. This includes developing improved sample collection and delivery mechanisms, according to Venkateswaran. "All of this has to be appropriately systemized."
He and others have already started working on PCR sample delivery. They recently received a $1 million Small Business Research and Innovation grant to support the second phase of a project that aims to develop a more effective system for pushing larger quantities of liquid containing sample into PCR tubes, which they hope to have ready for use in two years.
Ultimately, there is room for even more genomic analysis systems, Venkateswaran said, adding that NASA is pursuing a number of opportunities. For example, biodetection and biodefense company PositiveID partnered with the US Defense Threat Reduction Agency and NASA in 2014 to develop a handheld microfluidic PCR device, called Firefly Dx, for various uses including sampling the microbiome aboard the space station and soil sampling on Mars missions. The agency is also open to partnering with more companies who are interested in testing their technologies for potential use in space, he said, adding that there is no one-size-fits-all solution.
But there are some requirements. In 2013, researchers from Thermo Fisher Scientific's Ion Torrent tested their sequencing chips' tolerance to amounts of radiation equivalent to what they might encounter on a two-year Mars mission. They found that the irradiation did not affect the electrical performance of the chips and it did not have a measurable effect on sequencing quality. However, the sequencer's size is too big for space flight, which is why it has not been nominated for use, Venkateswaran said. Besides being lightweight, systems designed for space flight also have to consider fluidics and sample containment issues and use as few consumables as possible to avoid unnecessary waste, he said. They also have to be easy enough for astronauts to run, he added.