Single Cell Applications for Target Enrichment

General Background
The overall goal of this project will be the generation of a de novo mutation map in human sperm. This will be achieved by two key-objectives: 1.) establishment of single-cell targeted re-sequencing, and 2.) sperm metagenomics.

  • establishment of single-cell targeted re-sequencing
  • sperm metagenomics

Project description
This pilot project will focus on the following steps: 1.) establishing targeted resequencing technologies for single cells (combination of whole genome amplification methods with targeted re-sequencing), 2.) whole genome amplification (WGA) of sperm cells followed by targeted re-sequencing of 10-100 disease genes, with the final goal to do this for the whole exome.

De novo mutations are an increasingly recognized cause of rare and common sporadic diseases (Hoischen et al. Nat Genet 2010, 2011; Vissers et al. Nat Genet 2010). Great advancements to this end have been made utilizing several technological developments. The greatest impact has come through the broad application of exome sequencing, the first tenable genomewide method for the detection of point mutations. The relative low frequency of de novo mutations in the human exome (range of 0-4/individual) allows fast recognition of potential disease causing mutations. The systematic studies of parent-patient trios now allowed the systematic study of disease causing de novo mutations (Riviere, Bon, Hoischen et al. Nat Genet 2012; de Ligt et al. in preparation).
From individual disease studies it is known that the vast majority of de novo point mutations is transmitted via the paternal germcells, due to the higher rate of mitotic divisions of sperm compared to oocytes (23 mitotic divisions in woman, and e.g. 800 mitotic divisions in 50-year old males). This is only systematically studied for a small proportion of dominant sporadic diseases collectively described as "paternal age effect" (PAE) disorders (Goriely & Wilkie, AJHG 2012). Next to the higher mutation rates in males, several point mutations have been described to have a selective advantage in sperm (Goriely & Wilkie, AJHG 2012). To better understand the distribution of de novo mutation in the human exome/genome, we propose the following study: Generation of a de novo mutation map in human sperm. For this study a collaboration with the Department for Gynecology and Obstetrics was established, and sperm donor samples of 100 anonymous but healthy donors as well as DNA from blood of all donors is available. In addition, several other samples with underlying expected somatic mutations, that would ultimately benefit from single cell sequencing approaches are available via collaborations.

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