Microbe Detectives in Training: New Course Uses Novel Sequencing Technology

MinION Oxford Nanopore Technology

Millions of organisms, invisible to the blind eye, make up much of the world around us, and the world inside our bodies. These organisms are microbes, or microorganisms influential in the cause of disease, infection and fermentation.

Though 99.9% of microbes cannot currently be cultured in a lab – meaning they cannot be multiplied and studied using traditional methods – the emergence of genomic sequencing over the last two decades has revolutionized medicine and biology.

In recent years, the human microbiome has become a hot topic in the field of biology, as certain microbes have been linked to diabetes, obesity, depression and even autism. With the development of Sanger sequencing in 1977 – in which an electric current pushes a molecule through lanes of polymer gel, separating DNA fragments according to size – researchers began sequencing microbial genomes one base pair at a time. This expanded our understanding of the genetic identities, lifestyles, capabilities, toxicities and infectious natures of different microbes. With enough information, microbiologists have even identified contaminant-eliminating microbes that can help clean up oil spills and landfill waste.

Though the development of sequencing changed the field of microbiology, older techniques have proven to be painstakingly slow, as bacterial genomes consist of several million base pairs (not to mention the human genome, which is made up of 3.2 billion). While working on his doctorate 20 years ago, UVA associate professor of biology Martin Wu recalls only being able to collect 300 base pairs in a day.

Flash forward two decades, and Wu is now teaching a group of undergraduate students how to sequence hundreds of thousands of base pairs in a matter of seconds.

Titled “NextGen Sequencing: Minions the Microbe Detective,” his course utilizes MinIONs, an Oxford Nanopore Technology. At the size of a thumb drive, the device contains hundreds of parallel pores with dimensions of only a few nanometers. In essence, each MinION contains one membrane that acts like a barrier, and when proteins are placed into this membrane, they act as channels, allowing DNA base pairs to pass through. The four base pairs of DNA – adenine, cytosine, guanine and thymine – are different shapes and sizes, blocking the electric current in different ways. When the MinION picks up on the different signature changes in current, it is able to sequence each DNA fragment in real time.

By using the MinION sequencing technology, students were able to sequence the genome of a COVID-19 isolate to identify the specific variant it belongs to.

“[Researchers] can do the sequencing in the field, and before, no one could do that because the other technologies for DNA sequencing require bulky and very expensive equipment,” Wu said. “[The MinION] is cheap, it is mobile, it is in real-time and it is easy to use. That is why we are able to bring this to the classroom.”

Inspiration for the novel course came three years ago, when Wu conducted a demonstration in front of his microbiology class of 150 students, using the MinION device to sequence gut microbes in front of their eyes. In response to lots of excitement, Wu decided to develop a course for students to have the hands-on experience themselves.

By simply hooking a MinION up to any computer, the device can generate up to 50 billion bases of DNA sequencing. Individually priced at $1,000, MinION DNA sequencers fall within the lab’s budget, unlike older sequencing technologies which can cost between $100,000 and $500,000. Now, students are not only learning how to use the cutting-edge technology, but also how to prepare samples for sequencing.

Fourth-year biology major and computer science minor Simonne Guenette enrolled in the course for its unique lab experience. Guenette is currently working through a UVA biology lab, in partnership with a start-up biotech company, engineering E. coli to turn waste plastic into biodegradable plastic. She plans on applying to immunology and microbiology doctoral programs after graduation.

“The course is about taking samples of bacteria or a virus, which have hundreds or thousands of different species, and learning how to process and prepare that sample so that you can sequence the DNA of everything in it, and figure out what is in it,” Guenette said. “This can be really helpful for figuring out if there is any cause for concern, if there are any known pathogens in your sample, or if it is an indicator of good soil health,” for example.

In the first of two course projects, students posed their own questions regarding the species makeup of a sample of their choice. From saliva to soil health to kombucha, the variety of the projects highlighted the many dimensions in which microbiomes can be studied. Guenette, along with two other students, sequenced the microbes of a SCOBY, or symbiotic culture of bacteria and yeast, which is commonly used to form sour foods such as kombucha and kimchi. The group gathered their sample from Cobouchy, a local, small coffee kombucha-brewing business owned by a friend of Guenette’s.

“[My friend] had always wanted to know what is in her kombucha because a lot of commercial kombucha owners know what bacteria are in their bottles,” Guenette said. “Since most kombucha is made out of tea, and this was made with coffee, she was curious whether there was any difference in what she was going to see in it.”

After collecting liquid from Cobouchy’s SCOBY, the group utilized silica spin columns to extract the DNA, and then magnetic beads to purify it. They then sequenced the DNA through the MinION, and collected the sequences on a computer program.

Fourth-year biology and global public health double-major Rachel Roenicke was also part of the SCOBY-sequencing group. With plans of obtaining a doctorate in virology post-graduation, Roenicke is also working in UVA’s Granger Lab researching genetic expression as it relates to eye development and disease.

“The most rewarding part of this course has been being able to do our own independent projects using the skills that we learned in the beginning of the semester … instead of just following a protocol like in a lot of my other lab classes,” Roenicke said. “Dr. Wu also did an amazing job of making everyone feel comfortable with the techniques, so we were able to confidently conduct our own projects.”

In the second project of the semester, students are extracting the RNA from inactive COVID-19 samples and turning it back into DNA for sequencing in order to identify any variants.

I want students to get hands-on experience to appreciate how powerful DNA sequencing is, and how it has become an essential tool to study biology or medicine.”
Wu said.  “It is not something remote that they think they have nothing to do with.”

Made commercially available in 2015, MinIONs are being used in Antarctica for studies on biodiversity, in Africa for tracing infectious disease, the International Space Station for astronomical data collection, and thanks to Wu, now at UVA.

“I hope [students] can appreciate the power of cutting-edge technology,” Wu said. “[I also want students to] apply that technology … to deal with pressing issues of our society. There is actually a practical use to being a microbial detective.”

Later this month, Wu will gather at the Nanopore Community Meeting 2021 with scientists from around the world to present on the educational experience of the course.