290 research outputs found

    Discovery and genotyping of structural variation from long-read haploid genome sequence data

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    In an effort to more fully understand the full spectrum of human genetic variation, we generated deep single-molecule, real-time (SMRT) sequencing data from two haploid human genomes. By using an assembly-based approach (SMRT-SV), we systematically assessed each genome independently for structural variants (SVs) and indels resolving the sequence structure of 461,553 genetic variants from 2 bp to 28 kbp in length. We find that &gt;89% of these variants have been missed as part of analysis of the 1000 Genomes Project even after adjusting for more common variants (MAF &gt; 1%). We estimate that this theoretical human diploid differs by as much as ∼16 Mbp with respect to the human reference, with long-read sequencing data providing a fivefold increase in sensitivity for genetic variants ranging in size from 7 bp to 1 kbp compared with short-read sequence data. Although a large fraction of genetic variants were not detected by short-read approaches, once the alternate allele is sequence-resolved, we show that 61% of SVs can be genotyped in short-read sequence data sets with high accuracy. Uncoupling discovery from genotyping thus allows for the majority of this missed common variation to be genotyped in the human population. Interestingly, when we repeat SV detection on a pseudodiploid genome constructed in silico by merging the two haploids, we find that ∼59% of the heterozygous SVs are no longer detected by SMRT-SV. These results indicate that haploid resolution of long-read sequencing data will significantly increase sensitivity of SV detection.</jats:p

    Laminin-332 alters connexin profile, dye coupling and intercellular Ca(2+ )waves in ciliated tracheal epithelial cells

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    BACKGROUND: Tracheal epithelial cells are anchored to a dynamic basement membrane that contains a variety of extracellular matrix proteins including collagens and laminins. During development, wound repair and disease of the airway epithelium, significant changes in extracellular matrix proteins may directly affect cell migration, differentiation and events mediated by intercellular communication. We hypothesized that alterations in cell matrix, specifically type I collagen and laminin α3β3γ2 (LM-332) proteins within the matrix, directly affect intercellular communication in ciliated rabbit tracheal epithelial cells (RTEC). METHODS: Functional coupling of RTEC was monitored by microinjection of the negatively charged fluorescent dyes, Lucifer Yellow and Alexa 350, into ciliated RTEC grown on either a LM-332/collagen or collagen matrix. Coupling of physiologically significant molecules was evaluated by the mechanism and extent of propagated intercellular Ca(2+ )waves. Expression of connexin (Cx) mRNA and proteins were assayed by reverse transcriptase – polymerase chain reaction and immunocytochemistry, respectively. RESULTS: When compared to RTEC grown on collagen alone, RTEC grown on LM-332/collagen displayed a significant increase in dye transfer. Although mechanical stimulation of RTEC grown on either LM-332/collagen or collagen alone resulted in intercellular Ca(2+ )waves, the mechanism of transfer was dependent on matrix: RTEC grown on LM-332/collagen propagated Ca(2+)waves via extracellular purinergic signaling whereas RTEC grown on collagen used gap junctions. Comparison of RTEC grown on collagen or LM-332/collagen matrices revealed a reorganization of Cx26, Cx43 and Cx46 proteins. CONCLUSION: Alterations in airway basement membrane proteins such as LM-332 can induce connexin reorganizations and result in altered cellular communication mechanisms that could contribute to airway tissue function

    Resolving the complexity of the human genome using single-molecule sequencing

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    The human genome is arguably the most complete mammalian reference assembly, yet more than 160 euchromatic gaps remain and aspects of its structural variation remain poorly understood ten years after its completion. To identify missing sequence and genetic variation, here we sequence and analyse a haploid human genome (CHM1) using single-molecule, real-time DNA sequencing. We close or extend 55% of the remaining interstitial gaps in the human GRCh37 reference genome - 78% of which carried long runs of degenerate short tandem repeats, often several kilobases in length, embedded within (G+C)-rich genomic regions. We resolve the complete sequence of 26,079 euchromatic structural variants at the base-pair level, including inversions, complex insertions and long tracts of tandem repeats. Most have not been previously reported, with the greatest increases in sensitivity occurring for events less than 5 kilobases in size. Compared to the human reference, we find a significant insertional bias (3:1) in regions corresponding to complex insertions and long short tandem repeats. Our results suggest a greater complexity of the human genome in the form of variation of longer and more complex repetitive DNA that can now be largely resolved with the application of this longer-read sequencing technology

    Genomic mapping of phosphorothioates reveals partial modification of short consensus sequences

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    Bacterial phosphorothioate (PT) DNA modifications are incorporated by Dnd proteins A-E and often function with DndF-H as a restriction-modification (R-M) system, as in Escherichia coli B7A. However, bacteria such as Vibrio cyclitrophicus FF75 lack dndF-H, which points to other PT functions. Here we report two novel, orthogonal technologies to map PTs across the genomes of B7A and FF75 with >90% agreement: single molecule, real-time sequencing and deep sequencing of iodine-induced cleavage at PT (ICDS). In B7A, we detect PT on both strands of G[subscript ps]AAC/G[subscript ps]TTC motifs, but with only 12% of 40,701 possible sites modified. In contrast, PT in FF75 occurs as a single-strand modification at C[subscript ps]CA, again with only 14% of 160,541 sites modified. Single-molecule analysis indicates that modification could be partial at any particular genomic site even with active restriction by DndF-H, with direct interaction of modification proteins with GAAC/GTTC sites demonstrated with oligonucleotides. These results point to highly unusual target selection by PT-modification proteins and rule out known R-M mechanisms.National Natural Science Foundation (China)Ministry of Science and Technology of the People's Republic of China (973 and 863 Programs)Shanghai Municipal Council of Science and Technology. Shanghai Pujiang ProgramNational Science Foundation (U.S.) (CHE-1019990)National Institute of Environmental Health Sciences (ES002109)Singapore. National Research Foundation (Singapore-MIT Alliance for Research and Technology

    Chromosomal-level assembly of the Asian Seabass genome using long sequence reads and multi-layered scaffolding

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    We report here the ~670 Mb genome assembly of the Asian seabass (Lates calcarifer), a tropical marine teleost. We used long-read sequencing augmented by transcriptomics, optical and genetic mapping along with shared synteny from closely related fish species to derive a chromosome-level assembly with a contig N50 size over 1 Mb and scaffold N50 size over 25 Mb that span ~90% of the genome. The population structure of L. calcarifer species complex was analyzed by re-sequencing 61 individuals representing various regions across the species' native range. SNP analyses identified high levels of genetic diversity and confirmed earlier indications of a population stratification comprising three clades with signs of admixture apparent in the South-East Asian population. The quality of the Asian seabass genome assembly far exceeds that of any other fish species, and will serve as a new standard for fish genomics

    A random six-phase switch regulates pneumococcal virulence via global epigenetic changes

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    Streptococcus pneumoniae (the pneumococcus) is the world's foremost bacterial pathogen in both morbidity and mortality. Switching between phenotypic forms (or 'phases') that favour asymptomatic carriage or invasive disease was first reported in 1933. Here, we show that the underlying mechanism for such phase variation consists of genetic rearrangements in a Type I restriction-modification system (SpnD39III). The rearrangements generate six alternative specificities with distinct methylation patterns, as defined by single-molecule, real-time (SMRT) methylomics. The SpnD39III variants have distinct gene expression profiles. We demonstrate distinct virulence in experimental infection and in vivo selection for switching between SpnD39III variants. SpnD39III is ubiquitous in pneumococci, indicating an essential role in its biology. Future studies must recognize the potential for switching between these heretofore undetectable, differentiated pneumococcal subpopulations in vitro and in vivo. Similar systems exist in other bacterial genera, indicating the potential for broad exploitation of epigenetic gene regulation.Ana Sousa Manso, Melissa H. Chai, John M. Atack, Leonardo Furi, Megan De Ste Croix, Richard Haigh, Claudia Trappetti, Abiodun D. Ogunniyi, Lucy K. Shewell, Matthew Boitano, Tyson A. Clark, Jonas Korlach, Matthew Blades, Evgeny Mirkes, Alexander N. Gorban, James C. Paton, Michael P. Jennings, Marco R. Oggion

    Human CD34+ CD133+ Hematopoietic Stem Cells Cultured with Growth Factors Including Angptl5 Efficiently Engraft Adult NOD-SCID Il2rγ−/− (NSG) Mice

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    Increasing demand for human hematopoietic stem cells (HSCs) in clinical and research applications necessitates expansion of HSCs in vitro. Before these cells can be used they must be carefully evaluated to assess their stem cell activity. Here, we expanded cord blood CD34+ CD133+ cells in a defined medium containing angiopoietin like 5 and insulin-like growth factor binding protein 2 and evaluated the cells for stem cell activity in NOD-SCID Il2rg−/− (NSG) mice by multi-lineage engraftment, long term reconstitution, limiting dilution and serial reconstitution. The phenotype of expanded cells was characterized by flow cytometry during the course of expansion and following engraftment in mice. We show that the SCID repopulating activity resides in the CD34+ CD133+ fraction of expanded cells and that CD34+ CD133+ cell number correlates with SCID repopulating activity before and after culture. The expanded cells mediate long-term hematopoiesis and serial reconstitution in NSG mice. Furthermore, they efficiently reconstitute not only neonate but also adult NSG recipients, generating human blood cell populations similar to those reported in mice reconstituted with uncultured human HSCs. These findings suggest an expansion of long term HSCs in our culture and show that expression of CD34 and CD133 serves as a marker for HSC activity in human cord blood cell cultures. The ability to expand human HSCs in vitro should facilitate clinical use of HSCs and large-scale construction of humanized mice from the same donor for research applications.Singapore-MIT Alliance for Research and Technology ( Infectious Diseases research grant

    Serum after Autologous Transplantation Stimulates Proliferation and Expansion of Human Hematopoietic Progenitor Cells

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    Regeneration after hematopoietic stem cell transplantation (HSCT) depends on enormous activation of the stem cell pool. So far, it is hardly understood how these cells are recruited into proliferation and self-renewal. In this study, we have addressed the question if systemically released factors are involved in activation of hematopoietic stem and progenitor cells (HPC) after autologous HSCT. Serum was taken from patients before chemotherapy, during neutropenia and after hematopoietic recovery. Subsequently, it was used as supplement for in vitro culture of CD34+ cord blood HPC. Serum taken under hematopoietic stress (4 to 11 days after HSCT) significantly enhanced proliferation, maintained primitive immunophenotype (CD34+, CD133+, CD45−) for more cell divisions and increased colony forming units (CFU) as well as the number of cobblestone area-forming cells (CAFC). The stimulatory effect decays to normal levels after hematopoietic recovery (more than 2 weeks after HSCT). Chemokine profiling revealed a decline of several growth-factors during neutropenia, including platelet-derived growth factors PDGF-AA, PDGF-AB and PDGF-BB, whereas expression of monocyte chemotactic protein-1 (MCP-1) increased. These results demonstrate that systemically released factors play an important role for stimulation of hematopoietic regeneration after autologous HSCT. This feedback mechanism opens new perspectives for in vivo stimulation of the stem cell pool
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