KEY TAKEAWAYS:
- Research looked at potential of a hand-held gene sequencer that produces rapid results.
- Project used shotgun metagenomic sequencing, which essentially creates genetic fingerprints of all the organisms in a sample.
- Researchers compared hand held unit’s detection of pathogens in irrigation water to that of the gold standard Illumina sequencing technology
Referenced CPS research:
- Cooper 2021
When Kerry Cooper, Ph.D. with the University of Arizona, learned scientists had used a hand-held device in Africa to sequence genetic information from lowland gorillas, he wondered if it might be applicable to the produce industry. To that end, he is leading CPS-funded research to compare the cell phone-sized Oxford MinION genome sequencer to Illumina sequencing technology in detecting pathogens in agricultural water. “Why can’t we use this out in the field to get a more rapid response?” Cooper said about the MinION. “The produce industry wants the produce to get out as fast as possible because it’s a fresh product. This has the potential of providing a very rapid response — possibly in a couple of days or even faster as we develop it.”
Although the concept sounded promising, he said researchers first had to determine whether the portable unit produced results comparable to Illumina technology, considered the gold standard in sequencing. Joining Cooper as co-investigators in the project, titled “Microbial characterization of irrigation waters using rapid, inexpensive and portable next generation sequencing technologies,” were Kelly Bright, Ph.D.; Channah Rock, Ph.D.; and Walter Betancourt, Ph.D., all with the University of Arizona.
The research is tapping shotgun metagenomic sequencing, a relatively new technology. Instead of testing for genetic information specific to an individual microbe, an often labor-intensive and costly task, the shotgun approach essentially creates genetic fingerprints of all the organisms in a sample. “It takes an irrigation water sample and sequences all of the DNA that’s in there — all of the bacteria, fungi and viruses,” Cooper said. A computer program then matches the DNA sequences to a genetic database of known organisms for identification.
As part of the project, the researchers collected irrigation water samples from the Yuma and Maricopa production areas and spiked them with different quantities of three bacterial and two viral pathogens and one protozoan pathogen. They ran them though the MinION as well as the Illumina. The goal was to determine detection limits of the hand-held unit. It worked well in detecting bacterial pathogens, though Cooper noted that their spiking levels were still much higher than populations typically found in irrigation water.
“It’s a good starting point without enrichment straight out of the irrigation water,” he said. But the MinION did not perform as well detecting, which they used as a surrogate for Cyclospora and other protozoa. “We just need to come up with a better method to get the DNA,” he said. When it came to assaying for viruses, Cooper said the hand-held unit was not able to detect any DNA or RNA associated with them. He blamed it partly on the general physiology and make up of viruses that makes them more challenging to detect than bacteria or protozoa. Water turbidity also influenced sequencing success. If it was in the low to medium range, it didn’t cause issues. But Cooper said even the low turbidity levels used in the tests would be pushing the sediment limits that the produce industry typically uses.
In the high turbidity samples, they had to first drop out the sediments before sequencing. The researchers plan to continue to work with the MinION to try to reduce bacterial detection limits from what they initially saw. “We’re applying some different technologies to bring that down, maybe a couple more logs, to where you’re in real-world detection limits,” Cooper said. “We’re also working on ways to get the protozoan extracts that will work.”
Nevertheless, he said he believed the MinION holds promise for the produce industry. “It’s got a lot of potential, but we’re at just step one of probably four or five steps,” Cooper said. “We confirmed it will work potentially for the bacteria. Now it’s tweaking it to get it to work better.”