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MUSE Science Museum, University of Verona Create Mobile Genomics Lab with MinIon

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NEW YORK (GenomeWeb) – Demonstrating the potential of Oxford Nanopore Technologies' MinIon to be used in the field, the MUSE Science Museum in Trento, Italy and the University of Verona have created a mobile genomics laboratory — and plan to demonstrate that DNA extraction, amplification, and sequencing can be done from remote locations like the Tanzanian forest.

For their initial project, the group performed 16S sequencing of a Tanzanian frog species and sent the data via satellite back to MUSE Trento, potentially identifying a new species.

"This is a proof of principle to show that it is now possible to do real-time sequencing in the most remote areas of the planet, and in extreme environments," Massimo Delledonne, the scientific director of the Expedition Genomics Lab, told GenomeWeb.  

Delledonne said that the mobile laboratory is essentially three backpacks containing the necessary equipment for extraction, amplification, and sequencing.

In one bag there are pipettes, tubes, and other components used for DNA extraction, amplification, and quantification. Commercially available solutions are used for DNA extraction, while the PCR amplification and DNA quantification are performed with a proprietary device developed in collaboration with Biodiversa, which can be powered by solar panels. Amplification is performed with a ready-to-use PCR kit stabilized at room temperature.

A second bag contains two MinIon devices, a laptop, portable batteries, and solar panels. The third bag is a cooler that contains the kit for library preparation and sequencing flowcells. Delledonne said that although the library prep kit is typically stored at -20 degrees C, the team "found that they are stable for at least one week at 4 degrees C."

Delledonne added that in order to reduce the number of components that are typically stored at -20 degrees C, they amplified the barcoding region with phosphorylated primers and then used the sequencing kit. Essentially, that allowed them to skip two steps before ligating the motor protein to the DNA fragments — end repair and dA tailing of the DNA. Since they used PCR products, "the first step was not necessary because we did not fragment DNA," Delledonne said.

Delledonne said that the team used the most recent flowcell, F7.3 and version 5 of the chemistry.

Before carrying out sequencing in the field, the team tested its equipment and protocols at the MUSE tropical greenhouse to simulate the hot temperatures and high humidity the team would experience in Tanzania. The final tests were done without electricity to simulate "wild" conditions, Delledonne said.

Michele Menegon, the coordinator of the project and a researcher at MUSE, told GenomeWeb that one of the goals of the project is to help with conservation planning, which "requires accurate information on the actual biological and evolutionary value of species and areas, in order to prioritize investments."

Being able to sequence DNA directly in the field in order to identify species is important because in many of the regions with high amounts of biodiversity, like the tropics, there are few sequencing facilities nearby. "In situ sequencing will fill the time gap between the collection of the sample and the recovering of the relevant molecular information on it," she said. In addition, she said, it will allow the whole process of biodiversity assessment to take place in the same country.

Aside from sequencing the Tanzanian frog, the team plans to sequence species in other areas of the tropics, such as Borneo, and will "sequence various organisms belonging to other groups of vertebrates like reptiles and mammals in order to test performances across taxa," Menegon said.

No major modifications had to be made to the MinIon to enable it to sequence in the field, Delledonne said. However, Oxford Nanopore did enable the MinKnow software to run without an Internet connection and enabled Metrichor — the cloud-based basecalling software — to allow the team to upload raw reads even with a slow Internet connection to build the consensus sequence, he said.

Delledonne said that because the experiment was for the purposes of barcoding, only about 600 bp of DNA needed to be sequenced. That region was amplified using a freeze-dried kit from Biodiversa.

The frog that the team sequenced had a 95 percent identity with the leaf litter frog Arthroleptis xenodactyloides, and Delledonne said that the 5 percent difference is "way above the average species threshold within the genus," meaning that the species is a putative novel species. However, he noted that confirmation would need to be done via Sanger sequencing. Full results of the sequencing project will be presented at an Oxford Nanopore conference next week, Delledonne said.

"We are coming back with a very good set of data related to the functionality of the different components in such a difficult environment," he added.

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