Single-cell copy number variation detection

Detection of chromosomal aberrations from a single cell by array comparative genomic hybridization (single-cell array CGH), instead of from a population of cells, is an emerging technique. However, such detection is challenging because of the genome artifacts and the DNA amplification process inherent to the single cell approach. Current normalization algorithms result in inaccurate aberration detection for single-cell data. We propose a normalization method based on channel, genome composition and recurrent genome artifact corrections. We demonstrate that the proposed channel clone normalization significantly improves the copy number variation detection in both simulated and real single-cell array CGH data

New Array Approaches to Explore Single Cells Genomes

Microarray analysis enables the genome-wide detection of copy number variations and the investigation of chromosomal instability. Whereas array techniques have been well established for the analysis of unamplified DNA derived from many cells, it has been more challenging to enable the accurate analysis of single cell genomes. In this review, we provide an overview of single cell DNA amplification techniques, the different array approaches, and discuss their potential applications to study human embryos

Somatic Genomic Variations in Early Human Prenatal Development

Only 25 to 30% of conceptions result in a live birth. There is mounting evidence that the cause for this low fecundity is an extremely high incidence of chromosomal rearrangements occurring in the cleavage stage embryo. In this review, we gather all recent evidence for an extraordinary degree of mosaicisms in early embryogenesis. The presence of the rearrangements seen in the cleavage stage embryos can explain the origins of the placental mosaicisms seen during chorion villi sampling as well as the chromosomal anomalies seen in early miscarriages. Whereas these rearrangements often lead to implantation failure and early miscarriages, natural selection of the fittest cells in the embryo is the likely mechanism leading to healthy fetuses

Meiotic errors followed by two parallel postzygotic trisomy rescue events are a frequent cause of constitutional segmental mosaicism

Structural copy number variation (CNV) is a frequent cause of human variation and disease. Evidence is mounting that somatic acquired CNVs are prevalent, with mosaicisms of large segmental CNVs in blood found in up to one percent of both the healthy and patient populations. It is generally accepted that such constitutional mosaicisms are derived from postzygotic somatic mutations. However, few studies have tested this assumption. Here we determined the origin of CNVs which coexist with a normal cell line in nine individuals. We show that in 2/9 the CNV originated during meiosis. The existence of two cell lines with 46 chromosomes thus resulted from two parallel trisomy rescue events during postzygotic mitoses

Single-Cell RNA-Sequencing Reveals a Continuous Spectrum of Differentiation in Hematopoietic Cells.

The transcriptional programs that govern hematopoiesis have been investigated primarily by population-level analysis of hematopoietic stem and progenitor cells, which cannot reveal the continuous nature of the differentiation process. Here we applied single-cell RNA-sequencing to a population of hematopoietic cells in zebrafish as they undergo thrombocyte lineage commitment. By reconstructing their developmental chronology computationally, we were able to place each cell along a continuum from stem cell to mature cell, refining the traditional lineage tree. The progression of cells along this continuum is characterized by a highly coordinated transcriptional program, displaying simultaneous suppression of genes involved in cell proliferation and ribosomal biogenesis as the expression of lineage specific genes increases. Within this program, there is substantial heterogeneity in the expression of the key lineage regulators. Overall, the total number of genes expressed, as well as the total mRNA content of the cell, decreases as the cells undergo lineage commitment.The study was supported by Cancer Research UK grant number C45041/A14953 to A.C., C.L. and L.F and a core support grant from the Wellcome Trust and MRC to the Wellcome Trust–Medical Research Council Cambridge Stem Cell Institute. S.T would like to acknowledge the Lister Research Prize from the Lister Institute. The authors declare no competing financial interestsThis is the final version of the article. It first appeared from Cell Press via http://dx.doi.org/10.1016/j.celrep.2015.12.08

Development of a novel ozone- and photo-stable HyPer5 red fluorescent dye for array CGH and microarray gene expression analysis with consistent performance irrespective of environmental conditions

<p>Abstract</p> <p>Background</p> <p>Array-based comparative genomic hybridization (CGH) and gene expression profiling have become vital techniques for identifying molecular defects underlying genetic diseases. Regardless of the microarray platform, cyanine dyes (Cy3 and Cy5) are one of the most widely used fluorescent dye pairs for microarray analysis owing to their brightness and ease of incorporation, enabling high level of assay sensitivity. However, combining both dyes on arrays can become problematic during summer months when ozone levels rise to near 25 parts per billion (ppb). Under such conditions, Cy5 is known to rapidly degrade leading to loss of signal from either "homebrew" or commercial arrays. Cy5 can also suffer disproportionately from dye photobleaching resulting in distortion of (Cy5/Cy3) ratios used in copy number analysis. Our laboratory has been active in fluorescent dye research to find a suitable alternative to Cy5 that is stable to ozone and resistant to photo-bleaching. Here, we report on the development of such a dye, called HyPer5, and describe its' exceptional ozone and photostable properties on microarrays.</p> <p>Results</p> <p>Our results show HyPer5 signal to be stable to high ozone levels. Repeated exposure of mouse arrays hybridized with HyPer5-labeled cDNA to 300 ppb ozone at 5, 10 and 15 minute intervals resulted in no signal loss from the dye. In comparison, Cy5 arrays showed a dramatic 80% decrease in total signal during the same interval. Photobleaching experiments show HyPer5 to be resistant to light induced damage with 3- fold improvement in dye stability over Cy5. In high resolution array CGH experiments, HyPer5 is demonstrated to detect chromosomal aberrations at loci 2p21-16.3 and 15q26.3-26.2 from three patient sample using bacterial artificial chromosome (BAC) arrays. The photostability of HyPer5 is further documented by repeat array scanning without loss of detection. Additionally, HyPer5 arrays are shown to preserve sensitivity and data quality from gene expression experiments.</p> <p>Conclusion</p> <p>HyPer5 is a red fluorescent dye that behaves functionally similar to Cy5 except in stability to ozone and light. HyPer5 is demonstrated to be resistant to ozone at up to 300 ppb, levels significantly higher than commonly observed during summer months. Consequently, HyPer5 dye can be used in parallel with Cy3 under any environmental conditions in array experiments.</p

Mouse model of chromosome mosaicism reveals lineage-specific depletion of aneuploid cells and normal developmental potential.

Most human pre-implantation embryos are mosaics of euploid and aneuploid cells. To determine the fate of aneuploid cells and the developmental potential of mosaic embryos, here we generate a mouse model of chromosome mosaicism. By treating embryos with a spindle assembly checkpoint inhibitor during the four- to eight-cell division, we efficiently generate aneuploid cells, resulting in embryo death during peri-implantation development. Live-embryo imaging and single-cell tracking in chimeric embryos, containing aneuploid and euploid cells, reveal that the fate of aneuploid cells depends on lineage: aneuploid cells in the fetal lineage are eliminated by apoptosis, whereas those in the placental lineage show severe proliferative defects. Overall, the proportion of aneuploid cells is progressively depleted from the blastocyst stage onwards. Finally, we show that mosaic embryos have full developmental potential, provided they contain sufficient euploid cells, a finding of significance for the assessment of embryo vitality in the clinic.We acknowledge the Wellcome Trust for supporting this work. H.B was supported by a Wellcome Trust clinical PhD fellowship. We acknowledge the Research Foundation Flanders (FWO) (FWO-G.A093.11 and FWO-G.0924.15 to T.V.; the travel grant V.4.140.10.N.00 to T.V.; N.V.d.A., E.F.G. and K.T. are Ph.D. students supported by FWO 1.1.H.28.12, FWO G.0924.15 and FWO 1126016N, respectively) and KU Leuven SymBioSys (PFV/10/016 to T.V.; P.K. is a PhD. student supported with PFV/10/016).This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/ncomms1116

Self-assembly of embryonic and two extra-embryonic stem cell types into gastrulating embryo-like structures.

Embryonic stem cells can be incorporated into the developing embryo and its germ line, but, when cultured alone, their ability to generate embryonic structures is restricted. They can interact with trophoblast stem cells to generate structures that break symmetry and specify mesoderm, but their development is limited as the epithelial-mesenchymal transition of gastrulation cannot occur. Here, we describe a system that allows assembly of mouse embryonic, trophoblast and extra-embryonic endoderm stem cells into structures that acquire the embryo's architecture with all distinct embryonic and extra-embryonic compartments. Strikingly, such embryo-like structures develop to undertake the epithelial-mesenchymal transition, leading to mesoderm and then definitive endoderm specification. Spatial transcriptomic analyses demonstrate that these morphological transformations are underpinned by gene expression patterns characteristic of gastrulating embryos. This demonstrates the remarkable ability of three stem cell types to self-assemble in vitro into gastrulating embryo-like structures undertaking spatio-temporal events of the gastrulating mammalian embryo.European Research Council (669198) Wellcome Trust (098287/Z/12/Z)

Identification of region-specific astrocyte subtypes at single cell resolution

Astrocytes, a major cell type found throughout the central nervous system, have general roles in the modulation of synapse formation and synaptic transmission, blood-brain barrier formation, and regulation of blood flow, as well as metabolic support of other brain resident cells. Crucially, emerging evidence shows specific adaptations and astrocyte-encoded functions in regions, such as the spinal cord and cerebellum. To investigate the true extent of astrocyte molecular diversity across forebrain regions, we used single-cell RNA sequencing. Our analysis identifies five transcriptomically distinct astrocyte subtypes in adult mouse cortex and hippocampus. Validation of our data in situ reveals distinct spatial positioning of defined subtypes, reflecting the distribution of morphologically and physiologically distinct astrocyte populations. Our findings are evidence for specialized astrocyte subtypes between and within brain regions. The data are available through an online database (https://holt-sc.glialab.org/), providing a resource on which to base explorations of local astrocyte diversity and function in the brain.status: publishe

Defining the Design Principles of Skin Epidermis Postnatal Growth

Summary During embryonic and postnatal development, organs and tissues grow steadily to achieve their final size at the end of puberty. However, little is known about the cellular dynamics that mediate postnatal growth. By combining in vivo clonal lineage tracing, proliferation kinetics, single-cell transcriptomics, and in vitro micro-pattern experiments, we resolved the cellular dynamics taking place during postnatal skin epidermis expansion. Our data revealed that harmonious growth is engineered by a single population of developmental progenitors presenting a fixed fate imbalance of self-renewing divisions with an ever-decreasing proliferation rate. Single-cell RNA sequencing revealed that epidermal developmental progenitors form a more uniform population compared with adult stem and progenitor cells. Finally, we found that the spatial pattern of cell division orientation is dictated locally by the underlying collagen fiber orientation. Our results uncover a simple design principle of organ growth where progenitors and differentiated cells expand in harmony with their surrounding tissues.Peer reviewe