University of Vermont

College of Medicine

“Going Deep” at the VCC/COM Massively Parallel Sequencing Facility

Scott Tighe and Timothy Hunter in the Massively Parallel Sequencing Facility
From left, Scott Tighe, senior research technician, and Timothy Hunter, director of the Advanced Genome Technologies Core, in the Massively Parallel Sequencing Facility. (Photo by Raj Chawla, UVM Medical Photography)

The late astronomer Carl Sagan’s famous “billions and billions” quote seemed comical in the later 20th century, but no longer. Few people, even including science fiction geeks and biomedical scientists, would have ever imagined our world would have the technology to literally sequence billions – up to 350, in fact – nucleotide bases (all 25,000 genes) of DNA in eight days. That’s the capacity of massively parallel sequencing, a feature of the University of Vermont’s Advanced Genome Technologies Core and a relatively new facility supported by the Vermont Cancer Center, the Lake Champlain Cancer Research Organization, Inc., UVM College of Agriculture and Life Sciences, and UVM College of Medicine.

Run by Timothy Hunter, director, and Scott Tighe, senior research technician, the Massively Parallel Sequencing Facility is located in the College of Medicine’s Health Science Research Facility, adjacent to the Microarray Facility and DNA Analysis Lab. Launched in 2011 and established fully in 2012, the facility provides deep sequencing services for a number of UVM researchers, including Aimee Shen, Ph.D., assistant professor of microbiology and molecular genetics, Arti Shukla, Ph.D., associate professor of pathology, and Russell Tracy, Ph.D., professor of pathology and biochemistry.

According to information shared by Hunter, this expensive and time-intensive process is “the new ‘holy grail’ in cancer and other biomedical research.” The technology’s advantages include the ability to discover new mutations and alterations for drug targeting, determine epigenetic – non-DNA-related – interactions, discover new RNA transcripts, and detect unknown foreign DNA.

“I think massively parallel sequencing opens up a whole new world of unsolved research mysteries that may include disease process to drug effectiveness,” says Shukla.

Vermont Cancer Center Co-Directors Gary Stein, Ph.D., and Claire Verschraegen, M.D., describe the Massively Parallel Sequencing Facility’s capabilities as “spectacular.” In addition, shares Stein, the facility was the pivotal element in his and Vermont Cancer Center colleagues’ successful receipt of a $2.1 million Pfizer grant, which supports deep sequencing for establishing epigenetic signatures for the earliest stages of breast, uterine and ovarian cancer.

“We are confident that this advanced insight into the early warning signs of major cancers will be a cornerstone to the roadmap for prevention that is being developed by the Vermont Cancer Center for Vermonters and beyond,” Stein says.

While attractive due to its capabilities, the technology is not ideal for every research project. To determine which projects are best suited, Hunter and Tighe conduct mandatory consultations to ensure all perspectives are equally considered. At this consult, all key players involved in the workflow are seated at the same table, including, most importantly, Jeffrey Bond, Ph.D., and Julie Dragon, Ph.D., in the Molecular Bioinformatics Shared Resource.

“We hammer out such issues as experimental design, discuss expectations and timeframes,” says Hunter. “This leads to interactive discussion as the project continues.”

Investigators with approved projects drop off DNA, RNA, cell or tissue samples to either the refrigerator or freezer, which begins the process of creating an amplified DNA target library, says Tighe. The facility’s core management system – called iLab – provides notice of the sample’s arrival and an order form. Each sample must be fragmented into specific sizes, which is accomplished by using a Sonicator. The sample goes into a tubule; water circulates while acoustic waves shoot into it, and then a series of enzymatic manipulations take place. “These are used to create what is called a sequencing library, which is then used to bind to the heart of the sequencer – a flow cell,” says Tighe. The flow cell resembles a microscope slide with channels and portals.

“The sample needs to be as perfect as it can be before it goes on the flow cell, because they are so expensive,” says Tighe, who pays careful attention to quality control.

The slide features probes, and the fragments, says Hunter, and will find complements or match and fold over, amplifying and making another copy of the fragment, each of which has a primer bound to it.

“You can amplify DNA fragments over and over again,” says Tighe. “This creates hundreds of millions of clusters, each of which contains about1,000 identical copies of specific genome template molecule fragments.”

Finally, the flowcell moves to the Illumina HiSeq 1000, which is where the sequencing occurs. Portals at the top and bottom of the slide allow for exchange.

Tracy and colleagues, including Paula Tracy, Ph.D., professor of biochemistry, in the College’s coagulation and thrombosis research community, have worked closely with Hunter, Tighe, Bond and Dragon, as part of a large project.

“We wanted to sequence the complete genome of a research subject with a very rare condition – complete deficiency of a critical coagulation factor, Factor V,” Tracy says. He explains that the Factor V gene – called F5 – is a large gene with many similarities to the gene for Factor VIII, which is better known as the gene responsible for hemophilia A. Scientists believe complete deficiency of Factor V should be fatal, and surmise that anyone with a complete deficiency must have some other compensating change that prevents a fatal outcome. Tracy and his colleagues wanted to obtain a complete genomic sequence rather than just the F5 sequence.

“The analysis provided by Tim and Scott in the Core was superb with over 30X coverage (a measure of high quality) across the genome,” Tracy says. “Turnaround was timely, and the bioinformatics collaboration from Jeff and Julie has been critical to our work so far. To date, we’ve identified a damaging mutation from one of the subject’s parents, and are exploring the F5 gene to identify the other damaging mutation. After that, we’ll begin the work of trying to identify the compensating mutation. This work couldn’t have been done here at UVM without the facility and the people who make it work."

Hunter and Tighe have experienced other successful outcomes with additional faculty.

Shen says the capacity of the facility provided her lab with the ability to study a very critical area – the sporulation pathway in C. difficile – which resulted in an important paper due to publish in PLoS Genetics on August 8, 2013. “I can’t over-emphasize how much better it is to be able to hash through problems face-to-face and have the bioinformaticists really try and understand the science,” says Shen. “We ran into a number of unforeseen problems along the way, and Scott was able to really help us troubleshoot the issue due to his excellent connections in the sequencing field,” she adds.

The process has multiple benefits, but most importantly, says Hunter, “Each project is a relationship.”

Shukla sees the benefits of those relationships, too. “Every time I sit down with bioinformaticists to discuss my data, I am amazed by the unlimited possibilities and ways to analyze data and draw conclusions,” she says. “In my opinion, the addition of massively parallel sequencing to the College of Medicine is one of the best things that has ever happened to me as a researcher.”