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Although a cell produces an assortment of products that protect, inspect and if necessary heal its valuable genetic code, DNA-mutations can accumulate during a cell’s life cycle. This is exemplified by the acquisition of somatic genetic lesions that cause cancer, but also by the birth of disabled children due to a genomic aberration acquired during gametogenesis or early embryogenesis. In fact, most likely we all are genetic mosaics early on or later in our life, with part of our cells containing a genetic repertoire that deviates from the original zygotic genome. As a consequence, the ability to characterize the entire genome of a single human cell for all classes of genetic variation is important to unravel the extent, nature and consequences of this mosaicism, as well as to establish a better understanding of the underlying genomic instability during processes like gametogenesis , embryogenesis and tumorigenesis.Single-cell sequencing is thus an invaluable tool not only for basic genome research but also for enabling novel clinical applications (e.g. profiling circulating tumor cells from peripheral blood of a cancer patient to guide the patient’s treatment using ‘liquid biopsies’ of solid tumours). At the start of my Ph.D., single-cell sequencing did not exist. Before the genome of a single cell can be analyzed on current high-throughput sequencing platforms, its genome must be amplified thousands of times to obtain enough input material. This step of whole genome amplification (WGA) poses a tremendous challenge, as it delivers a biased representation of the original genome, containing amplification artifacts that resemble real genetic variants. In chapter 4 of this dissertation we developed a method for paired-end sequencing of single-cell genomes, which exploits paired-end mapping and single nucleotide variant information for sifting WGA-artifacts from true unbalanced copy number variants. In addition, we were able to demonstrate for the first time the detection of inter- and intra-chromosomal structural rearrangements in a single cell.In addition to WGA-bias, ongoing DNA-replication poses another challenge for single-cell genomics. A snapshot of a diploid cell in S-phase demonstrates consecutive loci of copy number state 2, 3 or 4. The number of these loci, their size and copy number state is dynamic over the entire S-phase. In chapter 3 of this dissertation we used single-cell array comparative genomic hybridization to demonstrate that such ongoing DNA-replication negatively impacts reliable copy number profiling of the cell. When analyzing cells randomly selected from a population and without knowing their cell cycle phase, this can lead to misinterpretation of the cell’s copy number profile. In chapter 5 we developed a methodology based on single-cell sequencing for the detection of S-phase genomes in a population of sequenced diploid cells, and allowing the emergence of the genome-wide DNA-replication program of a single cell at high resolution.Recently it has been found that the first cleavage divisions of human life following in vitro fertilization (IVF) are prone to chromosome instability (CIN). Several observations suggest that also in vivo human embryogenesis is affected by such instability. In chapter 6, we combined single-cell sequencing of all available blastomeres of human cleavage stage embryos with live-cell imaging of the embryos’ development since conception. Reading the genome of each cell at high resolution combined with valuable information about cell division, cell behavior and morphology delivered deeper insight in the operation of CIN during human embryogenesis. We discovered novel natures of chromosome rearrangement including interstitial (submicroscopic) copy number variants, as well as novel mechanisms of CIN including the absorption of a polar body by a blastomere with subsequent vast centric fission following division of this blastomere. In addition, we delivered unambiguous proof for the occurrence of breakage fusion bridge cycles during the first cell cycles of human life, a mechanism of chromosome rearrangement frequently observed in cancer. At the moment, genomic disorders and constitutional de novo DNA-rearrangements are mainly considered to result from pre-meiotic or meiotic germ line errors. However, depending on which blastomere(s) contribute(s) to the inner cell mass and embryo proper, this CIN during embryogenesis may be a source of not only pregnancy loss, but hypothetically also of genomic disorders and constitutional de novo structural variants (SVs), including de novo DNA copy number variants (CNVs).To gain insight in the evolutionary conservation of CIN during embryogenesis, we studied genome stability at different time-points of mouse embryogenesis in chapter 7. Unexpectedly, we found the genome of mouse embryos to be much more stable than observed in human embryos, providing a stepping-stone for further research towards the identification of the molecular mechanisms underlying CIN.