W289 RAPiD-Seq A novel approach to genotype by sequencing reduced genome representations

Date: Sunday, January 15, 2012
Time: 11:00 AM
Room: Sunrise
Marcio Resende Jr , University of Florida, Gainesville, FL
Leandro Gomide Neves , University of Florida, Gainesville, FL
Kelly Mayrink Balmant , University of Florida, Gainesville, FL
Christopher Dervinis , University of Florida, Gainesville, FL
Daleen VanDyk , University of Pretoria, Pretoria, South Africa
Alexander Myburg , Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa
Matias Kirst , University of Florida - SFRC, Gainesville, FL
With the rapid decrease in DNA analysis throughput and cost, direct sequencing has emerged as an option for genotyping, based on the detection of variants in sequencing reads. DNA polymorphism detection may be carried out by resequencing the entire genome of individuals of interest. But for most species, this approach is still too costly and the analysis too complex to be carried out in thousands of individuals. An increasingly popular method of sequencing/genotyping is to produce reduced genome representations by restriction enzyme digestion followed by adaptor ligation and PCR amplification, prior to sequencing and polymorphism detection. This approach requires several sample-specific enzymatic steps and large amounts of high quality DNA, and the number of fragments and the regions of the genome that can be sampled is limited by existing restriction enzymes. Here we report a simplified approach of genotyping (RAPiD-Seq) that eliminates restriction digestion and adaptor ligation. RAPiD-Seq relies on a two-step PCR-based enrichment of genome representations, followed by sequencing and polymorphism detection. We demonstrate the use of RAPiD-Seq in the sequencing and genotyping of over 30,000 40-nt fragments from 96 individuals of an Eucalyptus hybrid family, of which 9,000 are variable in the population. Cost of genotyping using RAPiD-Seq is an order of magnitude lower compared to commercial platforms, for a similar number of loci, and the technical simplicity allows one technician to process 480 samples daily using standard laboratory equipment. Development is currently underway for maize, loblolly pine and poplar.