For Keygene, the Dutch plant-breeding technology company, next-generation sequencing has been instrumental in developing faster and easier ways to pinpoint mutations and variations in plant genomes.
For the last 18 years, the company has been developing and applying new technologies for an international group of vegetable seed companies that back it financially. Over the last year, Keygene has used both 454 Life Sciences’ and Solexa’s next-gen sequencers to develop new research methods, banking on each system’s strength for different applications.
The two sequencing platforms complement each other well, according to Michiel van Eijk, manager for upstream research at Keygene. “For [marker] discovery purposes, where longer sequence reads are needed, the 454 system is necessary,” he said. “But for marker detection purposes, the much larger throughput and lower cost per base of the Solexa platform actually makes this affordable.”
Last week, Keygene presented some of its projects at the Plant and Animal Genome conference in San Diego (see Table in last week’s issue).
KeyPoints and CRoPS
The company last week launched a method for detecting mutations in mutagenized plant libraries. The technology, called KeyPoint, involves sequencing of PCR products on 454’s Genome Sequencer to pinpoint mutations in genes of interest. KeyPoint, according to the company, improves and replaces the current TILLING (“targeting induced local lesions in genomes”) method, which detects chemically induced mismatch mutations using an enzyme.
While the enzyme-based approach requires researchers to confirm mutations by Sanger sequencing afterwards, KeyPoint detects mutations and sequences the region around them all in one go. “Basically, it skips one step of the procedure,” said van Eijk. “It’s very fast and it’s very accurate, because it is based on highly redundant sequencing.”
The company is now offering KeyPoint as a service to its customers, and van Eijk expects it will be cost-competitive with the TILLING method. “With the  FLX coming, with the higher throughput, the cost will be hard to beat,” he said. Keygene, which has had a GS 20 in house for one year, expects to receive its GS FLX by early March.
KeyPoint is not Keygene’s first application involving next-generation sequencing. For years scientists have been looking for new polymorphisms in plants by amplifying portions of the genome, and sequencing them using the Sanger method. Keygene developed this amplification-based approach, called Amplified Fragment Length Polymorphism, or AFLP. A year ago, the company introduced a new technology, called CRoPS for complexity reduction of polymorphic sequences, that uses 454 sequencing to sequence the fragments generated by AFLP.
“The limiting step was the cost of sequencing,” van Eijk said. “When 454 came around, that basically enabled us to do what we had been hoping to do for many years already.”
Over the last year, KeyGene has developed an automated bioinformatics pipeline to process data coming out of CRoPS experiments, which it presented at last week’s PAG meeting. “Now with the push of a button, we can process data from one or several 454 runs, which gives us a list of putative SNPs.” The next step is to convert these SNPs into a SNP assay for genotyping, he added, and Keygene presented results on this last week as well.
Keygene also recently adopted 454 sequencing to determine the position of transposons in transposon insertion libraries, and offers this technique as a service now. Last week, it presented results from a petunia library project, a collaboration with a group at Radboud University in Nijmegen in the Netherlands.
“The limiting step was the cost of sequencing. When 454 came around, that basically enabled us to do what we had been hoping to do for many years already.”
Transposon insertion libraries are used for functional genomics studies, and being able to determine where the transposons are located, van Eijk claimed, “transforms the way that plant geneticists can work with these populations.”
In the past, researchers used a modified AFLP approach to detect when a transposon entered a specific gene — “a relatively low-throughput technology,” van Eijk said. “Now we can sequence the entire collection and put it into a database.”
A few weeks ago Keygene completed a pilot experiment in collaboration with Solexa, in which it sequenced AFLP fragments to genotype plants, and presented the findings at PAG last week. In contrast to CRoPS, which is used to discover polymorphisms, this method is used for marker detection — which is why the shorter reads of Solexa’s Genetic Analyzer are sufficient.
Up until now, researchers ran the AFLP fragments out on gels and compared their sizes. That step is now replaced by high-throughput sequencing.
“If the read length is long enough to distinguish the AFLP fragment from other ones, then there is no point in having longer reads,” van Eijk said. “The reason why we do this with the Solexa platform is that is has many more reads [per run].”
For the pilot project, which involved 125 markers and 95 samples, Keygene generated the amplified fragments and sent them to Solexa, for sequencing. The next steps will be to scale up and optimize the technology, van Eijk said, and to explore whether it can detect heterozygotes.
Keygene, which is currently thinking about getting its own Solexa instrument, hopes to finish developing the method within six months, and to offer it as a service later this year.
While the company is “convinced of the ability” of Solexa’s system, he said, it will not ignore other next-generation sequencing platforms coming along. “With the near-availability of the Solexa platform, and the throughput and relatively low cost that it has, we tried this [platform] first to jump-start sequence-based AFLP detection,” van Eijk said. “Obviously, if in the future even lower-cost or higher-throughput platforms will come around, we are interested in that, at least to consider it.”