Keygene has used 454 Life Sciences’ Genome Sequencer 20 to help develop a technology platform that will enable large-scale SNP discovery and detection in higher eukaryotic organisms.
“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,” says Michiel van Eijk, manager of upstream research at Keygene.
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 five- to 10-fold redundancy with 454’s instrument. A typical sequence run yields more than 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 then applies quality measures to separate sequence errors from true polymorphisms, based on redundant sequencing, sample origin information, and allele frequencies.
“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,” comments Mark van Haaren, business development manager of Keygene. “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.”
The methods and apparatus concern sequencing single molecules of single-stranded DNA or RNA by exposing the molecule to exonuclease activity, removing free nucleotides one at a time from one end of the nucleic acid, and identifying the released nucleotides by Raman spectroscopy or FRET.
The patent covers a simplified strategy for sequencing large genomes. Clone-Array Pooled Shotgun Sequencing is based upon pooling rows and columns of arrayed genomic clones for shotgun library construction. Random sequences are accumulated and the data are assembled by sequential comparison of rows and columns, to resolve the sequence of clones at points of intersection. Compared to either a clone-by-clone approach or whole genome shotgun sequencing, CAPSS requires relatively few library constructions and only minimal computational power for a complete genome assembly.