The folding of genomic DNA from the beads-on-a-string like structure of nucleosomes into higher order assemblies is critically linked to nuclear processes. We have calculated the first 3D structures of entire mammalian genomes using data from a new chromosome conformation capture procedure that allows us to first image and then process single cells. This has allowed us to study genome folding down to a scale of <100 kb and to validate the structures. We show that the structures of individual topological-associated domains and loops vary very substantially from cell-to-cell. By contrast, A/B compartments, lamin-associated domains and active enhancers/promoters are organized in a consistent way on a genome-wide basis in every cell, suggesting that they could drive chromosome and genome folding. Through studying pluripotency factor- and NuRD-regulated genes, we illustrate how single cell genome structure determination provides a novel approach for investigating biological processes.
The success of genome-wide association studies has paralleled the development of efficient genotyping technologies. We describe the development of a next-generation microarray based on the new highly-efficient Affymetrix Axiom genotyping technology that we are using to genotype individuals of European ancestry from the Kaiser Permanente Research Program on Genes, Environment and Health (RPGEH). The array contains 674,517 SNPs, and provides excellent genome-wide as well as gene-based and candidate-SNP coverage. Coverage was calculated using an approach based on imputation and cross validation. Preliminary results for the first 80,301 saliva-derived DNA samples from the RPGEH demonstrate very high quality genotypes, with sample success rates above 94% and over 98% of successful samples having SNP call rates exceeding 98%. At steady state, we have produced 462 million genotypes per week for each Axiom system. The new array provides a valuable addition to the repertoire of tools for large scale genome-wide association studies.
The Kaiser Permanente (KP) Research Program on Genes, Environment and Health (RPGEH), in collaboration with the University of California-San Francisco, undertook genome-wide genotyping of .100,000 subjects that constitute the Genetic Epidemiology Research on Adult Health and Aging (GERA) cohort. The project, which generated .70 billion genotypes, represents the first large-scale use of the Affymetrix Axiom Genotyping Solution. Because genotyping took place over a short 14-month period, creating a near-real-time analysis pipeline for experimental assay quality control and final optimized analyses was critical. Because of the multi-ethnic nature of the cohort, four different ethnic-specific arrays were employed to enhance genome-wide coverage. All assays were performed on DNA extracted from saliva samples. To improve sample call rates and significantly increase genotype concordance, we partitioned the cohort into disjoint packages of plates with similar assay contexts. Using strict QC criteria, the overall genotyping success rate was 103,067 of 109,837 samples assayed (93.8%), with a range of 92.1-95.4% for the four different arrays. Similarly, the SNP genotyping success rate ranged from 98.1 to 99.4% across the four arrays, the variation depending mostly on how many SNPs were included as single copy vs. double copy on a particular array. The high quality and large scale of genotype data created on this cohort, in conjunction with comprehensive longitudinal data from the KP electronic health records of participants, will enable a broad range of highly powered genome-wide association studies on a diversity of traits and conditions. KEYWORDS genome-wide genotyping; GERA cohort; Affymetrix Axiom; saliva DNA; quality control T HE Genetic Epidemiology Research on Adult Health and Aging (GERA) resource is a cohort of .100,000 subjects who are participants in the Kaiser Permanente Medical Care Plan, Northern California Region (KPNC), Research Program on Genes, Environment and Health (RPGEH) (detailed description of the cohort and study design can be found in dbGaP, Study Accession: phs000674.v1.p1). Genome-wide genotyping was targeted for this cohort to enable large-scale genome-wide association studies by linkage to comprehensive longitudinal clinical data derived from extensive KPNC electronic health record databases. The cohort is multi-ethnic, with 20% minority representation (African American, East Asian, and Latino or mixed), and the remaining 80% nonHispanic white. For this project, four ethnic-specific arrays were designed based on the Affymetrix Axiom Genotyping System (Hoffmann et al. 2011a,b). The genotyping assay experiment took place over a 14-month period and to our knowledge, is the single largest genotyping experiment to date, producing .70 billion genotypes. The magnitude of the experiment, in conjunction with the long duration and simultaneous high throughput, required new protocols for assuring quality control (QC) during the assays and new genotyping strategies in postassay data analysis.Samp...
Phlebopus portentosus is a well-known edible wild mushroom in the tropical part of Yunnan province of China. The mushrooms grow around natural or planted trees of Delonix regia, Mangifera indica, Coffea arabica, Citrus grandis, Artocarpus heterophyllus and Quercus spp. The technology of cultivation of P. portentosus was developed and its biotrophic status examined at Xishuangbanna, Yunnan, China. Uncultivated red soils with and without host plants of C. arabica, C. grandis and M. indica were inoculated with solid inocula of P. portentosus. Matured mushrooms were produced from both inoculated soils, with and without the host plants, 20-30 days after inoculation. No mycorrhizal structures were detected although the fungal mycelia colonized the plant root surfaces. Results show that P. portentosus is a saprobic rather than a symbiotic fungus. Based on this discovery, two methods of cultivation of P. portentosus were developed. Non-sterilized agricultural soils in polypropylene bottles or bags were inoculated with the fungal solid inocula and incubated at a mushroom house. Fruiting-body primordia were produced from the inoculated soils 20-30 days after inoculation. Soil-cased sawdust logs inoculated with P. portentosus produced primordia 10-15 days after casing only. The primordia developed into mature mushrooms 5-6 days later with weights ranging from 20.0 to 135.0 g. The identity of the cultivated fruiting bodies was confirmed by morphological and molecular methods. Our molecular phylogeny based on the Internal Transcribed Spacer sequences from our cultivated isolate and Genbank accessions provides preliminary insight into the phylogeogrpahy of P. portentosus.
We aimed to identify genomic traits of transitions to ectomycorrhizal ecology within the Boletales by comparing the genomes of 21 symbiotrophic species with their saprotrophic brown-rot relatives.Gene duplication rate is constant along the backbone of Boletales phylogeny with large loss events in several lineages, while gene family expansion sharply increased in the late Miocene, mostly in the Boletaceae.Ectomycorrhizal Boletales have a reduced set of plant cell-wall-degrading enzymes (PCWDEs) compared with their brown-rot relatives. However, the various lineages retain distinct sets of PCWDEs, suggesting that, over their evolutionary history, symbiotic Boletales have become functionally diverse. A smaller PCWDE repertoire was found in Sclerodermatineae. The gene repertoire of several lignocellulose oxidoreductases (e.g. laccases) is similar in brown-rot and ectomycorrhizal species, suggesting that symbiotic Boletales are capable of mild lignocellulose decomposition. Transposable element (TE) proliferation contributed to the higher evolutionary rate of genes encoding effector-like small secreted proteins, proteases, and lipases. On the other hand, we showed that the loss of secreted CAZymes was not related to TE activity but to DNA decay.This study provides novel insights on our understanding of the mechanisms influencing the evolutionary diversification of symbiotic boletes.
Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs), inducing acute pyelonephritis and may result in permanent renal scarring and failure. Alpha-hemolysin (HlyA), a key UPEC toxin, causes serious tissue damage; however, the mechanism through which HlyA induces kidney injury remains unclear. In the present study, granulocyte-macrophage colony-stimulating factor (GM-CSF) secreted by renal epithelial cells was upregulated by HlyA in vitro and in vivo, which induced M1 macrophage accumulation in kidney, and ADAM10 was found involved in HlyA-induced GM-CSF. Macrophage elimination or GM-CSF neutralization protected against acute kidney injury in mice, and increased GM-CSF was detected in urine of patients infected by hlyA-positive UPEC. In addition, HlyA was found to promote UPEC invasion into renal epithelial cells by interacting with Nectin-2 in vitro. However, HlyA did not affect bacterial titers during acute kidney infections, and HlyA-induced invasion did not contribute to GM-CSF upregulation in vitro, which indicate that HlyA-induced GM-CSF is independent of bacteria invasion. The role of GM-CSF in HlyA-mediated kidney injury may lead to novel strategies to treat acute pyelonephritis.
Determining the long-range haplotypes in a diploid individual is a major technical challenge. Here we report a method of molecular haplotyping by directly imaging multiple polymorphic sites on individual human DNA molecules simultaneously. We demonstrate the utility of this technology by accurately determining the haplotypes consisting of up to 16 single-nucleotide polymorphisms in genomic regions up to 50 kilobases.The recent successful completion of the HapMap project 1 and the availability of low-cost, large-scale, high-throughput single-nucleotide polymorphism (SNP) genotyping platforms have paved the way for comprehensive genetic analysis of complex traits such as susceptibility to common diseases and drug responses through genome-wide association studies 2 . Haplotype analysis can enhance the statistical power in the mapping of human complex trait loci, with the potential of reducing the size of genome-wide association studies 3 . So far, the methods for determining haplotypes have considerable limitations that have prevented their use in large-scale and high-throughput genetic studies 4-7 .We had recently reported a DNA bar-coding method based on direct imaging of multiple polymorphic or sequence-motif sites on individual DNA molecules simultaneously [8][9][10] . Here we report a new molecular haplotyping approach, which improves the labeling efficiency and allows one to experimentally determine haplotypes of targeted genomic regions. Our method starts with long-range PCR amplification of target DNA segments containing the polymorphic sites followed by allele-specific labeling of polymorphic alleles with fluorescent dye molecules. The labeled double-stranded DNA (dsDNA) molecules are then stretched into linear form on a glass surface and imaged using multicolor total internal reflection fluorescence microscopy, a technique capable of localizing single fluorescent dye molecules with nanometer-scale accuracy 11 . By determining the order of the fluorescent labels on the backbone at all the polymorphic sites of fully labeled dsDNA fragments, the haplotype(s) of an individual can be inferred with great accuracy, in a manner similar to reading a bar code.Highly efficient allele-specific labeling of the markers on the long-range PCR products for single-molecule analysis is the most critical aspect of our haplotyping method. To increase the labeling efficiency, we generated single-stranded DNA (ssDNA) fragments from doublestranded long-range PCR products. We accomplished this by modifying one PCR primer with a 5′ Cy3 label and the other with 5′ phosphorylation in a PCR. When we incubated the resulting PCR product with lambda exonuclease, the DNA strand with the phosphorylated 5′ end was digested efficiently whereas the one with Cy3-modified 5′ end was protected from digestion and became the ssDNA template. We also used the end Cy3 label to orient the DNA molecules. Using this approach, we generated single ssDNA molecules up to 18 kilobases (kb) routinely. With ssDNA templates, the allele-specific extensi...
Fluorescence imaging and chromosome conformation capture assays such as Hi-C are key tools for studying genome organization. However, traditionally, they have been carried out independently, making integration of the two types of data difficult to perform. By trapping individual cell nuclei inside a well of a 384-well glass-bottom plate with an agarose pad, we have established a protocol that allows both fluorescence imaging and Hi-C processing to be carried out on the same single cell. The protocol identifies 30,000-100,000 chromosome contacts per single haploid genome in parallel with fluorescence images. Contacts can be used to calculate intact genome structures to better than 100-kb resolution, which can then be directly compared with the images. Preparation of 20 single-cell Hi-C libraries using this protocol takes 5 d of bench work by researchers experienced in molecular biology techniques. Image acquisition and analysis require basic understanding of fluorescence microscopy, and some bioinformatics knowledge is required to run the sequence-processing tools described here.
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