O-36: Genome Haplotyping and Detection of Meiotic Homologous Recombination Sites in Single Cells, A Generic Method for Preimplantation Genetic Diagnosis

Background: Haplotyping is invaluable not only to identify genetic variants underlying a disease or trait, but also to study evolution and population history as well as meiotic and mitotic recombination processes. Current genome-wide haplotyping methods rely on genomic DNA that is extracted from a large number of cells. Thus far random allele drop out and preferential amplification artifacts of single-cell whole genome amplifications as well as algorithmic shortcomings have precluded genome- wide haplotyping of one cell. Materials and Methods: Cells were isolated and lysed. The single-cell genomes were subsequently amplified using MDA- or PicoPlex technology, and SNPs were typed using Affymetrix or Illumina platforms. Genomewide haplotype reconstruction was performed with the Merlin as well as a newly developed family-based haplotyping algorithm PROSPER (preimplantation optimal screener and proper estimator of recombination). The reconstructed haplotypes were further interpreted using novel interpretation algorithms. Results: By developing an innovative data extraction and analysis pipeline of genome-wide SNP-data, the haplotype was reconstructed from single EBV-transformed lymphoblastoid cells as well as human blastomeres derived from in vitro fertilized (IVF) embryos. The methodology applies an optimized single-cell genotype computation and a mixture of novel algorithmic approaches that compute and interpret single-cell haplotypes. When compared to the reference haplotypes which were determined from DNA-samples extracted from many cells of the corresponding EBV cell lines, this computational pipeline improved genome-wide SNP-haplotype discordance rates from ~25% to less than 5% for single cells, thus enabling the detection of homologous recombination sites in individual cells. Furthermore, when applied to single human blastomeres of an embryo carrying a disease allele identified by conventional preimplantation genetic diagnosis the method was able to confirm the diagnosis. Conclusion: This generic haplotyping method will revolutionize reproductive genetic options as it not only enables to select pre-implantation embryos for a single trait, but also broadens the selection spectrum to multiple Mendelian traits as well as to qualitative and quantitative traits present in (animal) embryos. In addition, we envision that the method will increase our insights in the mechanisms of meiotic and mitotic recombination processes and enable the analysis of specimen with limited or degraded DNA.