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Ing. René Hogers works at KeyGene, a private research company that develops innovative applications and provides genetic analysis services for plant breeding groups in an effort to speed up the process of selecting plants with desirable traits. Currently, breeders are required to grow several generations to confirm the presence or absence of a particular phenotype. This can take years and waste precious resources on plants that will end up holding no value to the grower. To minimize this generational breeding process, Ing. Hogers is using Illumina's technology to enable breeders to realize significant savings while producing highly desirable agricultural products.

Illumina solutions for marker-assisted selection

Using VeraCode technology with the BeadXpress Reader, Ing. Hogers works with his customers to screen for genetically fit plants, a practice known as marker-assisted selection. Plants carrying the SNPs of interest are identified, allowing researchers to "decide which of the plants they should take and go on with and which ones they can throw away when the plants are still very small and with a just a few leaves. Picking out the right plants to go with the breeding processes saves a lot of time and also space in the greenhouses." For now, Ing. René Hogers has applied this technique to fruit and vegetable crops such as tomato, melon, pepper, lettuce, and cucumber, as well as field crops such as maize, barley, and potato.

Faster more, effective way to evaluate crops

Ing. René Hogers chose the BeadXpress Reader for several reasons, including the throughput. "We can screen for over 200 selected markers that will be informative, even for closely related species or parental lines, in one assay," he states. "This saves us time and costs." When asked about the BeadXpress workflow, Ing. Hogers replies, "It's more or less plug and play. With the availability of the [VeraCode] GoldenGate Assay, it will be easier to perform the assays. If we have a 384-plex [SNP assay], it takes about two to two-and-a-half hours to scan a whole plate of 96 samples." In addition, Ing. Hogers is pleased with the results. "The quality is quite good, it's very reproducible."

Excerpted from the September 2008, iCommunity article "Is Genetic Testing the Next Revolution in Agriculture?"

Dr. Richard Crooijmans is working to overcome new farming challenges. As a molecular geneticist at Wageningen University, he applies VeraCode technology and the BeadXpress Reader to genetic studies involving over 20,000 chickens and pigs. Many of these chicken and pig screenings are performed in an effort to remove a particular allele from the population, or as a way to study inheritance patterns. After analysis, only the desired animals are selected for breeding. To do this, Dr. Crooijmans needed a straightforward, accessible system.

Improving animal husbandry using Illumina technology

"We chose the VeraCode technology because it's pretty easy to use and everything is digital. You don't have to go through PCR where you have to load gels, with the potential to make mistakes. You don't have to check all the genotypes and enter them into a computer, where you can make mistakes again. With the VeraCode system, it's there and it's ready." A second reason for choosing VeraCode technology was the flexibility. "We have several regions we want to fine map, and [with VeraCode technology] we can just take specific SNPs and type them. This makes it very flexible. With other systems you have to do rather complicated assays and it also costs you more," he states. He sums up his experience by saying that VeraCode technology was "the easiest of the systems" he evaluated. When screening a large number of animals, the savings in time, cost, and effort add up significantly.

Faster more, cost-effective screening

With Dr. Crooijmans' efforts, SNPs associated with specific traits can be quickly and easily identified, eliminating the need for generational breeding. For example, a broiler chicken (one meant for consumption) has different desirable genetic traits than one destined for egg laying duties (a layer chicken). "Current commercial testing looks for a phenotype. It costs a large amount of money every time. You have to phenotype quite a lot of individuals and then screen them, and follow in time. We want to replace these very expensive phenotyping systems," states Dr. Crooijmans. If these can be replaced with faster, cost-effective molecular screening tests, the quality of the livestock population can be improved at a relatively rapid pace with minimal time and expense. When asked what the BeadXpress system has given him, Dr. Crooijmans answers, "knowledge and proven reliability as a partner in the animal sciences community. We showed that we can create and handle large data sets. With the help of commercial breeding partners, we have access to a wide range of animal material. Now we can get things done much more easily. Many of the grant proposals we submitted have been approved."

Excerpted from the September 2008, iCommunity article "Is Genetic Testing the Next Revolution in Agriculture?"

A molecular biologist by training, Dr. Martien Groenen has been studying complex traits in animals for more than 20 years at Wageningen University. He started out scanning the genome for microsatellites, looking for genetic markers associated with traits of interest. When Illumina's GoldenGate Genotyping panels hit the market, he quickly adopted these high-multiplex, high-throughput arrays for his research. Pleased with Illumina's technology, Groenen and collaborators decided to stay with Illumina to develop the first whole-genome genotyping array for the pig through the iSelect program.

i: What was your experience designing iSelect BeadChips?

For the PorcineSNP60 BeadChip, my group took a leading role in identifying a large number of SNPs through sequencing. In four months, using the Genome Analyzer, we identified 350,000 SNPs in the porcine genome. Within that same time frame we selected 70,000 and submitted them to be synthesized by Illumina. The results were great, 59,000 SNPs worked. I presented the design of the chip at the 2009 Plant and Animal Genome conference, and for the first time several groups heard the results. Everybody was really enthusiastic, which was very nice to hear.

And more or less we have done the same in chicken, but faster. We were involved in designing a 60K iSelect BeadChip in chicken. In this case, there were already three million known SNPs in the chicken genome. But we still used the Genome Analyzer to specifically identify new SNPs and to calculate the minor allele frequencies in the breeds that we wanted to use later on. This allowed us to have the maximum information content of the SNPs that were put on the chip. Once we had our sequence data, the whole process took six to seven weeks: analyzing the data, selecting the SNPs, and then submitting our SNP list to Illumina.

i: Why did you decide to go with Illumina for custom arrays?

Well, we began using Illumina's GoldenGate Assay a couple of years ago. We have continued using Illumina's technologies for two very important reasons. One is the reliability of the data and the quality of the data. The other thing is also the contact with Illumina itself, working with people at Illumina who have helped us get the results that we want. It's not just that you buy a product and that's it. You feel that for Illumina [the final product] is something that they want to bring to a good end.

i: Where do you see your research heading in the next few years?

The Genome Analyzer and iSelect, these different tools, are allowing much more rapid progress in agricultural research. Talking to my colleagues, we are very excited about these technologies, that's for sure. Looking ahead it's clear that at some point we will be completely resequencing individuals and I think we can expect a lot of information coming out of that.

If you would have asked me this question two or three years ago, what do you think you will be doing two or three years from now, I wouldn't have expected to do 60,000 SNPs on thousands of individuals or producing hundreds of millions of sequences in these species. Looking ahead for a couple of years and seeing developments in the field it's almost beyond imagining what is possible. I'm extremely excited working in this field and really in this period of time.

Excerpted from the September 2008, iCommunity article "Is Genetic Testing the Next Revolution in Agriculture?"

John McEwan is trying to improve the pastoral industry—specifically grazing animals and the products that come from them. As a member of the International Sheep Genomics Consortium, McEwan played a leading role in the development of the iSelect OvineSNP50 BeadChip, the first whole-genome genotyping array for sheep. His work primarily focuses on increasing the rate of genetic gain of sheep. To that end, he is currently attempting to identify a broad set of informative DNA markers for the sheep industry.

i: What was your strategy for designing a whole-genome array without a draft genome?

We just didn't have the money and we didn't have the time to sequence the sheep de novo, so we jumped onto next-generation sequencing technologies. We had a strategy where we used a long-read next-gen technology to get lumps of ovine sequence. We assembled those reads against the bovine genome to order and orientate the ovine contigs. Then we used the Illumina Genome Analyzer with the shorter reads and reduced representational library sequencing to provide the depth to find SNPs.

Doing this, we discovered more than 600,000 SNPs. We had about 300,000 good ones that passed the Illumina Infinium Assay design process. From these, we selected 60,000, and most of those made it onto the final OvineSNP50 BeadChip. A key point was that the assembly process allowed us to identify the SNP order and spacing, which greatly helped the quality of the final product.

Our goal in the sheep industry, is to move from the current situation of a having just a few DNA markers that can predict only a small proportion of the variation in performance to where we've got hundreds or thousands of markers and a much better prediction of the animal performance. With the Ovine BeadChip, people can now go directly from BeadChip results to markers that are immediately useful in the industry.

i: Will this strategy work for others in the agriculture community?

I think that vertebrate genomes are not the most complicated. Probably some of these large multi-ploid plant genomes will be more difficult. Within our institution there are a number of people who work with different plant species that are quite keen on the approach we took. I think with the current next-generation sequencing technology you should be able to quite rapidly and cost-effectively go from no sequence at all to an Illumina BeadChip. If you wanted to really push it, you could probably do it in a year and you could probably find all the SNPs required for less than several hundred thousand dollars.

i: How has the OvineSNP50 BeadChip performed?

I'm primarily interested in breeding sheep that can better resist diseases and, in particular, parasites. So we've genotyped 3,000 sheep, mainly sires, with the chip. Most of these sires have been evaluated for between approximately 40 to 200 progeny each for parasite resistance, growth, and various other traits. We got over 97% of the animals that passed the genotyping quality threshold on the first pass through and finally achieved 98.6% after rerunning a few samples. And we've got well over 99% genotype calls on the samples that passed the quality threshold. The performance of the BeadChip is really good. We're really happy about that.

i: What was you're experience working with Illumina to develop the OvineSNP50 BeadChip?

You obviously look over the fence at what other groups are doing and the experiences they are having and I think it would be fair to say that the experience in the bovine community was very good with Illumina, and the bovine community evaluated a number of platforms. Internally, our research group has also used a number of SNP genotyping technologies. I think Illumina has got a good, robust product and it's at a very good price.

We developed this BeadChip through a consortium. The great benefit of consortia is that people can do things that they couldn't do by themselves. Another hidden benefit is that there are many more people involved, each with different points of view. Often what finally comes out of the mix is actually a better solution. But, of course, that means all these people have to work together and communication becomes a very big issue.

This is where the Illumina staff comes in, particularly for the mechanical arrangements and the financial arrangements. They gave a lot of support to the community in getting the product out. The final product is much better because it involves animals from around the world, but it's also good for Illumina because they can sell a product that can be used anywhere in the world. The other benefit is technical advice. Obviously, Illumina works with a lot of other species. They've been down this path before and they can give you a lot of help and support in what they think is right, as well as what has worked well previously.