Specifically, the technology, called Complexity Reduction of Polymorphic Sequences, or CRoPS, will make large-scale polymorphism discovery more affordable in organisms with low levels of germplasm polymorphism and/or highly repeated genomes, which includes many crop species, the Dutch company said.
CRoPS, which Keygene launched during the Plant & Animal Genomes conference, held this week in San Diego, is evidence that next-generation sequencers could have a niche in the genotyping market.
The company used 454 Life Sciences' Genome Sequencer 20 to help it develop the CRoPS platform. "The 454 technology, in combination with Keygene's current technology platform, creates many new opportunities that further increase the efficiency of genotyping in plant species, of which CRoPS technology is the first example," Michiel van Eijk, manager of upstream research at Keygene, said in a statement.
For CRoPS, Keygene researchers use the company's AFLP technology to prepare tagged complexity-reduced libraries of two or more genetically diverse samples, which are then sequenced at 5- to 10-fold redundancy with 454's instrument. A typical sequence run yields over 200,000 sequence reads with a median length of 100 bases. The resulting sequences are clustered and bioinformatics tools are used to inspect the sequence contigs for differences.
Keygene scientists then apply quality measures to separate sequence errors from true polymorphisms, based on redundant sequencing, sample origin information, and allele frequencies. At the PAG meeting, Keygene is scheduled to present the results of SNP and simple sequence repeat mining and validation in pepper and maize.
"On pepper, a typical crop for which only very limited sequence information is available, we identified ... roughly 6,000 high quality polymorphic sites and hundreds of SSR sequences in only two CRoPS runs on the GS20 generating 450,000 reads," Mark van Haaren, business development manager of Keygene, told GenomeWeb News. This experiment wouldn't have been done using conventional Sanger sequencing because it's too expensive, he said.
"Basically, we are comparing sequences from two different samples, and we can directly identify the polymorphisms in those sequences that we generate with the 454 instrument," van Haaren said. "Up until now, we have been looking at fingerprints in bands on a gel - no sequence information whatsoever. With this new technology ... you can actually take each of the bands and sequence them. The fact that you can do many thousands of reads at the same time opens up this possibility."
Keygene hooked up with 454 roughly a year ago after approaching them following a GS20 presentation at a scientific meeting. Since then, the company has been sending samples to 454 to be sequenced. "We came up with a number of new applications for their technology in combination with our technology in the plant breeding field that gave us good results," van Haaren said. Last week Keygene announced it had purchased a Genome Sequencer 20.
Van Haaren said the company considered other next-generation sequencing vendors, including Solexa, which has reported a read length of 25 bases. Keygene chose 454 because the Branford, Conn.-based company was furthest along in implementing its technology, had already provided the company with good data, and promised to work on improving read length. "For us, the longer the read length, the better," van Haaren said.
Keygene plans to partner with 454 and use its own SNPWave technology, a multiplexed technology capable of detecting various subsets of sequences, including SNPs, to develop other applications in the field of plant genetics.
While next-generation sequencers can be used with other platforms to enhance genotyping --- which is what happened with CRoPS --- the instruments can also replace certain technologies. "Data quality per cost" is the key factor over which sequencing and genotyping compete, Jeff Schloss, program director of technology development at the National Human Genome Research Institute, told GenomeWeb News sister publication Pharmacogenomics Reporter in October.
Van Haaren said his company's SNPWave technology can look at between 10 and 100 or so SNPs at the same time, which is only a limited number. For some applications, you need more SNPs. If you calculate the cost, at a certain point, the 454 technology becomes of more interest.
Kate O'Rourke covers the next-generation genome-sequencing market for GenomeWeb News. E-mail her at [email protected].