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VBI's Otto Folkerts on the Promise of the GS FLX for Next-Gen Applications


Otto Folkerts
Associate Director of Technology Development
Virginia Bioinformatics Institute at Virginia Tech
Name: Otto Folkerts
Title: Associate Director of Technology Development, Virginia Bioinformatics Institute at Virginia Tech, since 2005
Age: 47
Experience: Various positions in business development and management of research collaborations at CuraGen, 1997-2005
Various research and managerial positions at Dow AgroSciences, 1989-1997
Master’s Degree in Enterprise Management, Lally School of Business, Rensselaer Polytechnic Institute, 2005
PhD in Plant Molecular Biology, Cornell University, 1989
BA and MS in Molecular and Cellular Biology from Agricultural University, Wageningen, the Netherlands, 1983 and 1985

Last week, the Virginia Bioinformatics Institute announced that it was among the first customers for the Genome Sequencer FLX from Roche Applied Science and 454 Life Sciences. VBI has installed the GS FLX in its Core Laboratory Facility alongside other high-throughput analysis platforms for sequencing, genotyping, gene expression analysis, and proteomics.
Otto Folkerts, associate director of technology development at VBI, said in a statement that the institute was “particularly excited” about new sequencing applications that the GS-FLX should enable, such as transcriptome sequencing, sequencing of clinical samples, and paired-end amplicon sequencing.
In Sequence spoke to Folkerts last week to discuss VBI’s plans for the new system.
When did your Genome Sequencer FLX arrive?
We had it installed [at our core facility] at the end of December. We have done some training runs, and it seems to be working just fine. We are looking at doing a couple of more practice runs, then we will open it up for projects with our own investigators here at VBI or collaborators across campus at Virginia Tech, or any other persons who want to access the technology.
To give you some background, VBI does bioinformatics research, but we also have wet labs — we have 16 research faculty with their own programs and over 200 employees. We have a large core lab facility that does high-throughput genomics, transcript profiling, and some proteomics research for customers — mostly academic researchers but also some small businesses. We have some large government customers as well.
What role does sequencing play at the core lab facility?
Most of the sequencing that we do at this point is ABI sequencing [on an ABI 3730 and an ABI 3100]. These are small projects, construct sequencing, amplicon sequencing, and that is predominantly a local business. We also have a very thriving Affymetrix platform that runs around the clock. We felt that with the advent of next-generation sequencing technologies, the cost for large sequencing projects is going to come down, and we would need to have a platform like that to complement our ongoing activities.
How did you decide to acquire a 454 Genome Sequencer?
We have done a number of projects with the 454 technology already. About a year ago, we sequenced the genome of Brucella abortus strain S19, which is a bacterium that is a category B biodefense agent. The genome sequencing was done at 454 through their measurement center. They provided the data back to us and we cleaned it up and closed the gaps with our ABI platform. We have a complete genome, 100 percent coverage, for the S19 strain as a result of that. We are finishing the publication as we speak.
The B. abortus strains that have been sequenced to date are the wildtype virulent pathogen, and the strain that we sequenced was the vaccine strain. By comparing the vaccine strains with the virulent strains, you get an insight into what attenuates the pathogen, and maybe this gives you new avenues into research to improve vaccine strains or come up with new vaccine strategies.
The second project we did with the 454 technology was Helicobacter pylori, a strain provided by a collaborator in Puerto Rico. The investigator is interested in understanding what is different between western European/North American strains and strains found in indigenous populations. It turns out there are very dramatic differences in H. pylori genomes, depending on where they come from. We completed that genome as well with 454 technology, and we are working on finishing the genome sequence. It was a little bit more challenging than the first one because of the particular nature of the genome.
Sequencing bacterial genomes and other pathogens fits well both with our institute’s mission to study infectious disease and the host-pathogen-environment interaction, as well as contracts we have with the National Institute of Allergy and Infectious Diseases and the Department of Defense in the biodefense area. As we are interested in doing more genome sequencing for biodefense, our own research interests are served by having a next-generation sequencing instrument. And because our experience with 454 was very positive, we felt that that was the best way to go.
We are aware that other next-generation technologies are being developed, but certainly not at the level of perfection that we would want to have in our laboratory.
We made the decision to purchase the instrument last summer. It took us a little while to get the funding together. The funding was provided by a grant from the State of Virginia that’s called the Commonwealth Research Initiative.
What projects will the new instrument mostly be used for?
Initially it will be mostly microbial genome sequencing. A couple of people in our institute have expressed an interest in doing this, and we have a few projects lined up in the next three months. We have already received one or two requests from the outside; people have heard through the grapevine that we have an instrument. All of those will be genome sequencing projects. But we have already started talking about other applications using the 454 platform. People have made inquiries about transcriptome sequencing. As Roche is rolling out new applications, supporting them with protocols, we will look at those applications as well, paired-end sequencing, amplicon sequencing. I am sure that there will be large numbers of customers.
When did you first become aware of next-generation sequencing?
I and one of my colleagues here in the bioinformatics group, Oswald Crasta, used to work for CuraGen [the majority-owner of 454 Life Sciences]. We both kind of witnessed the birth of the 454 technology. When I was at CuraGen [in charge of pharmaceutical collaborations and business development], one of the first projects that I did with 454 was a transcriptome sequencing project for an outside client. This was done before the GS 20 launched, so it was a very early implementation of the technology on a prototype instrument. And the results were just really incredible. Without doing anything fancy, no normalization of a library, just straightforward sequencing of double-stranded cDNA fragments, we got extremely nice coverage of the transcriptome that we wanted to sequence. That was for me the first illustration of the power of the technology. Up to that point, I might have been somewhat skeptical, ‘Do we need another sequencing technology? What can we get out of this?’ but now I am totally devoted.
How will the sequencing interact with bioinformatics at VBI?
I think there are two things that make this a really good fit for us. One is that we have the core lab, where we do routinely ABI sequencing. When people come to us and say, ‘We want to complete a genome’, we can do the gap closing for them, we have the bioinformatics infrastructure to figure out what the gaps are, and we can make primers and run the PCR and do standard ABI sequencing to close the gaps.
The other reason is that we have a really large cyberinfrastructure group, the group that we do most of our bioinformatics work in and that is providing the services to NIH and DoD under contract. That cyberinfrastructure group has tools and know-how and expertise to do genome analysis and annotation. So one of the other areas where we hopefully will bring in some customers is, instead of just providing the raw data, we can provide finished genome sequences and annotated genomes using the tools that we have built up in house and the expertise and the personnel we have. So for those investigators who want to have a fully finished, completely annotated genome, we can provide a single point of service, soup to nuts.
Did you have to develop some special bioinformatics tools to process the 454 data?
No. We have learned through our first two projects with 454 that the contig information that we get out using their assembly software is of high quality, so we don’t really need to do much there. Once we have a completed genome, gaps closed, we can use our already existing genome annotation pipeline to annotate the genome without having to do any custom bioinformatics work.
I think where [custom tools] might be appropriate is, as you go to larger genomes and you do de novo sequencing, the presence of repeated DNA sequences is going to be a problem for the 454 technology, and then you will need to rely on cloning and standard ABI sequencing to cover the DNA regions that have highly repetitive DNA sequences, and then you need to be able to combine that with the 454 sequence, while maintaining the quality scores. That’s probably where it becomes really tricky. But for bacterial genomes, I think, that’s less of an issue.
How are you planning to fund new sequencing projects?
The two projects I told you about earlier were funded out of our own funds. We will now look for new funding for more genome sequencing projects. If there is a good fit with some of the existing programs, we will try to take advantage of that. But my guess is, most of the money for our contracts is already allocated to specific projects, so any new sequencing we will do with new funds, and we will aggressively pursue the funding opportunities.
It may not be a competitive advantage to have a [454] instrument in house to get funding. We are aware of that. You have to be able to offer the lowest price. But I think most of the customers that I envision we will be working with in the first year or so are people who already have money and need to use it. And even though there is a considerable number of GS 20 machines out there and there is a large number of GS FLX machines that is going to be installed, there is a huge demand for the technology that exceeds the current capacity. It’s more cost-effective [than Sanger sequencing], and it’s just very trendy right now. As the costs are coming down, people are thinking of projects they can do with the technology – projects that they perhaps wanted to do for many years but which were just not affordable. Funding allocated for genome projects through NSF and NIH, it’s highly competitive, and it’s difficult to get a program funded. Even though you have a good idea and a very good research program that does not guarantee that you get the money for it. But now with the 454 technology, it’s going to be a lot more affordable to sequence such genomes.

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