62 research outputs found
Whole genome sequencing increases molecular diagnostic yield compared with current diagnostic testing for inherited retinal disease
No full textAbstract not availableJamie M. Ellingford, Stephanie Barton, Sanjeev Bhaskar, Simon G. Williams, Panagiotis I. Sergouniotis, James O, Sullivan, Janine A. Lamb, Rahat Perveen, Georgina Hall, William G. Newman, Paul N. Bishop, Stephen A. Roberts, Rick Leach, Rick Tearle, Stuart Bayliss, Simon C. Ramsden, Andrea H. Nemeth, Graeme C.M. Blac
Whole genome sequences are required to fully resolve the linkage disequilibrium structure of human populations
No full textBACKGROUND: An understanding of linkage disequilibrium (LD) structures in the human genome underpins much of medical genetics and provides a basis for disease gene mapping and investigating biological mechanisms such as recombination and selection. Whole genome sequencing (WGS) provides the opportunity to determine LD structures at maximal resolution.RESULTS: We compare LD maps constructed from WGS data with LD maps produced from the array-based HapMap dataset, for representative European and African populations. WGS provides up to 5.7-fold greater SNP density than array-based data and achieves much greater resolution of LD structure, allowing for identification of up to 2.8-fold more regions of intense recombination. The absence of ascertainment bias in variant genotyping improves the population representativeness of the WGS maps, and highlights the extent of uncaptured variation using array genotyping methodologies. The complete capture of LD patterns using WGS allows for higher genome-wide association study (GWAS) power compared to array-based GWAS, with WGS also allowing for the analysis of rare variation. The impact of marker ascertainment issues in arrays has been greatest for Sub-Saharan African populations where larger sample sizes and substantially higher marker densities are required to fully resolve the LD structure.CONCLUSIONS: WGS provides the best possible resource for LD mapping due to the maximal marker density and lack of ascertainment bias. WGS LD maps provide a rich resource for medical and population genetics studies. The increasing availability of WGS data for large populations will allow for improved research utilising LD, such as GWAS and recombination biology studies.<br/
The distribution of runs of homozygosity in the genome of river and swamp buffaloes reveals a history of adaptation, migration and crossbred events
No full textInternational audienceAbstractBackgroundWater buffalo is one of the most important livestock species in the world. Two types of water buffalo exist: river buffalo (Bubalus bubalis bubalis) and swamp buffalo (Bubalus bubalis carabanensis). The buffalo genome has been recently sequenced, and thus a new 90 K single nucleotide polymorphism (SNP) bead chip has been developed. In this study, we investigated the genomic population structure and the level of inbreeding of 185 river and 153 swamp buffaloes using runs of homozygosity (ROH). Analyses were carried out jointly and separately for the two buffalo types.ResultsThe SNP bead chip detected in swamp about one-third of the SNPs identified in the river type. In total, 18,116 ROH were detected in the combined data set (17,784 SNPs), and 16,251 of these were unique. ROH were present in both buffalo types mostly detected (~ 59%) in swamp buffalo. The number of ROH per animal was larger and genomic inbreeding was higher in swamp than river buffalo. In the separated datasets (46,891 and 17,690 SNPs for river and swamp type, respectively), 19,760 and 10,581 ROH were found in river and swamp, respectively. The genes that map to the ROH islands are associated with the adaptation to the environment, fitness traits and reproduction.ConclusionsAnalysis of ROH features in the genome of the two water buffalo types allowed their genomic characterization and highlighted differences between buffalo types and between breeds. A large ROH island on chromosome 2 was shared between river and swamp buffaloes and contained genes that are involved in environmental adaptation and reproduction
Heat shock protein expression is upregulated after acute heat exposure in three species of Australian desert birds
No full textDesert birds must cope with occasional and unpredictable heat waves, which are slowly becoming more frequent with climate change. Different orders of birds have different physiological and behavioural capacities that may aid survival during a heat wave. To date, the expression of genes related to heat exposure have not been studied across different bird orders. We hypothesised that acutely exposing native Australian birds whose natural habitat include arid environments to a high temperature (45 °C), similar to during a heat wave, would result in the upregulation of genes with protective effects against cell damage (BCL-2, VEGFA and heat shock proteins) and inflammation (interleukins), as well as the downregulation of genes involved in the coagulation pathway (fibrinogen). We used eight each of captive-bred Budgerigars (Melopsittacus undulatus), Zebra Finches (Taeniopygia guttata) and Diamond Doves (Geopelia cuneata). Four birds of each species were exposed to a temperature that was within the zone of thermal neutrality (35 °C), while the other four birds were exposed to a higher temperature (45 °C). The mRNA expression of selected genes were then measured using high-throughput qPCR platform (Fluidigm®, BioMark™). The results supported the hypothesis that acute exposure to a high temperature would result in the upregulation of heat shock protein (HSP) genes, but there was no significant upregulation of other genes with protective effects against cell damage nor genes associated with inflammation. The results also do not support the hypothesis that acute heat exposure would result in downregulation of the genes involved in the coagulation pathway in these birds. Among all the tissues that were analysed, the gastrointestinal tissue had the highest number of upregulated HSP genes, possibly indicating that this tissue requires the most protection to continue functioning. Diamond Dove organs also had the highest number of HSP genes upregulated, possibly a reflection of their ability to better protect their cells at high temperatures.Shangzhe Xie, Rick Tearle and Todd J. McWhorte
Three-dimensional regulation: Establishing novel linkages between non-coding genetic variation and target genes
No full textIn the human genome, 98% of the DNA is in non-gene coding regions. While these regions do not express genes, a mounting number of studies have shown that they are crucial to the maintenance of chromosome structure and in the regulation of gene expression. Although large epigenomics projects were established to functionally annotate non-coding regions, the comprehensive linkages between these regions and their target genes remain unknown. The human genome folds into hierarchical three-dimensional (3D) structure, bringing distantly regulatory elements into close proximity, leading to the formation of 3D chromatin physical interactions and playing an important role in the complex gene regulation network. Using chromatin interaction information, we can connect functional non-coding regions to their target genes to reveal novel regulation mechanisms. In Chapter 1, we reviewed current existing approaches to prioritise functional interactions from Hi-C data, the state-of-the-art data type used to study chromatin interactions, and categorised them into three classifications, including structuralbased methods, statistical model-based methods and data integration methods. Chapter 2 described the computational procedures of analysing Hi-C datasets, and introduced: HiC-QC, a tool that extracting summary statistics to perform quality control with Hi-C libraires; HiC-interactionmap and integration-tracks plot, tools to offer visualisation for Hi-C data integration. Additionally, aligners BWA and Bowtie2, were compared for their performance of mapping Hi-C data. Using type 1 diabetes (T1D) and regulatory T cells (Treg) as a disease-cell type model, based on data integration of Treg-specific Hi-C interactions and other epigenomics information, Chapter 3 established a filtering workflow called 3DFAACT-SNPs to link genetic variants that are associated with T1D to the loss of immune tolerance in Treg. Using this workflow, we identified 36 SNPs with plausible Treg-specific mechanisms of action contributing to T1D, linking 119 novel interacting regions. We demonstrated that it is possible to prioritise SNPs that contribute to disease based on regulatory function and illustrate the power of using chromatin interactions to connect non-coding SNPs to disease mechanisms. Lastly, Chapters 4 and 5 launch the statistically significant interaction profiling of 51 human cell lines and primary tissues from 173 public Hi-C datasets using a statistical model from MaxHiC, followed by investigating the uniqueness, distancing preference and the associated genes of the cell/tissue-specific interactions. We also identified interaction “hot zones”, regions with chromatin interactions observed across many cells and tissues. Using global and local enrichment analysis and a comparison to frequent interacting regions, we demonstrated the structural and regulatory functionality of the hot zones. We further comprehensively annotated chromatin interactions into 66 interaction classes, cataloguing potentially regulatory functional interactions for different cells and tissues. Finally, we revealed cell/tissue-specific 3D regulatory regions that are enriched with super-enhancers and overlapped with expression quantitative trait loci (eQTLs). Overall, using data integration and statistical models to prioritise functional chromatin interactions, this work produced novel computational tools and pipelines and generated valuable resource for the investigation of genome structure, demonstrating the power of using chromatin interactions to discover novel mechanisms in the genome and revealing novel linkages between noncoding DNA to traits/diseases.Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 202
Developmental timing of mutations revealed by whole-genome sequencing of twins with acute lymphoblastic leukemia
No full textAcute lymphoblastic leukemia (ALL) is the major pediatric cancer. At diagnosis, the developmental timing of mutations contributing critically to clonal diversification and selection can be buried in the leukemia's covert natural history. Concordance of ALL in monozygotic, monochorionic twins is a consequence of intraplacental spread of an initiated preleukemic clone. Studying monozygotic twins with ALL provides a unique means of uncovering the timeline of mutations contributing to clonal evolution, pre- and postnatally. We sequenced the whole genomes of leukemic cells from two twin pairs with ALL to comprehensively characterize acquired somatic mutations in ALL, elucidating the developmental timing of all genetic lesions. Shared, prenatal, coding-region single-nucleotide variants were limited to the putative initiating lesions. All other nonsynonymous single-nucleotide variants were distinct between tumors and, therefore, secondary and postnatal. These changes occurred in a background of noncoding mutational changes that were almost entirely discordant in twin pairs and likely passenger mutations acquired during leukemic cell proliferation.Yussanne Maa, Sara E. Dobbins, Amy L. Sherbornea, Daniel Chubb, Marta Galbiati, Giovanni Cazzaniga, Concetta Micalizzi, Rick Tearle, Amy L. Lloyd, Richard Hain, Mel Greaves, and Richard S. Houlsto
Metal resistant bacteria on gold particles: Implications of how anthropogenic contaminants could affect natural gold biogeochemical cycling
No full textIn Earth's near-surface environments, gold biogeochemical cycling involves gold dissolution and precipitation processes, which are partly attributed to bacteria. These biogeochemical processes as well as abrasion (via physical transport) are known to act upon gold particles, thereby resulting in particle transformation including the development of pure secondary gold and altered morphology, respectively. While previous studies have inferred gold biogeochemical cycling from gold particles obtained from natural environments, little is known about how metal contamination in an environment could impact this cycle. Therefore, this study aims to infer how potentially toxic metal contaminants could affect the structure and chemistry of gold particles and therefore the biogeochemical cycling of gold. In doing so, river sediments and gold particles from the De Kaap Valley, South Africa, were analysed using both microanalytical and molecular techniques. Of the metal contaminants detected in the sediment, mercury can chemically interact with gold particles thereby directly altering particle morphology and “erasing” textural evidence indicative of particle transformation. Other metal contaminants (including mercury) indirectly affect gold cycling by exerting a selective pressure on bacteria living on the surface of gold particles. Particles harbouring gold-tolerant bacteria with diverse metal resistant genes, such as Arthrobacter sp. and Pseudomonas sp., contained nearly two times more secondary gold relative to particles harbouring bacteria with less gold-tolerance. In conclusion, metal contaminants can have a direct or indirect effect on gold biogeochemical cycling in natural environments impacted by anthropogenic activity.Santonu Kumar Sanyal, Joël Brugger, Barbara Etschmann, Stephen M. Pederson, P.W. Jaco Delport, Roger Dixon, Rick Tearle, Alastair Ludington, Frank Reith, Jeremiah Shuste
Transcriptome profiling of infected chickens with newly emerged genotype VI I Newcastle disease virus strains
No full textSince it was reported in Indonesia in 1926, Newcastle disease (ND) is an endemic disease in Indonesia resulting in devastating economic losses in poultry. Despite heavy vaccination programs, NDV infection still occurs among commercial chicken farms, including vaccinated birds in Southeast Asia, especially in Indonesia. Therefore, the first objective was to detect and perform full genome sequencing of highly virulent circulating NDVs in vaccinated chicken flocks to identify the most adequate NDV strains as vaccine candidates. Our full genome sequencing analysis on two selected isolates from vaccinated birds in Indonesia has shown that both of them belong to highly virulent NDV-GVII.2 strains, while only 0.4% of the vaccine strains used in this country are genotype VII. Moreover, sequencing analysis of the existing genotype VII vaccine revealed that it has significant differences from GI vaccine strain, which is not sufficiently representative of genotype VII viruses. This finding illustrates that virulent NDV-GVII.2 strains are mainly responsible for the high morbidity and mortality of recent ND outbreaks in poultry in Indonesia. Virulent NDV-GVII strains were previously characterised as immunopathological phenotypes triggering severe tissue damage probably through apoptosis and necrosis of lymphoid tissues. However, the underlying molecular mechanism of pathogenesis of ND remains to be fully understood. Hence, the second objective was to identify and characterise the molecular mechanism of lymphotropic behaviour of NDV-GVII by focusing on detecting key biomarkers and cellular immune response signalling pathways that contribute to severe lymphocyte destruction in infected birds using RNA sequencing, bioinformatics tools and PPI network analysis. Transcriptomic profiling indicates that virulent NDV-GVII significantly downregulates immunologically regulated genes and innate immune regulating pathways including fMLP signalling in neutrophils, Fcy receptor-mediated phagocytosis in macrophages and monocytes, and leukocyte extravasation signalling and NF-kB activation by viruses. These transcriptional changes may lead to widespread immunosuppression and enhanced replication of the virus. As a result, the host’s immune response may be diminished, delayed, incomplete or display overly strong induction that can cause severe tissue damage. This finding also implies that virulent NDV-GVII strains appear to possess the capability to inhibit the induction of immune responses by targeting lymphocytes and destroying these cells, which may be one indispensable factor of the pathogenesis of NDV-GVII. Moreover, PPI network analysis revealed that the top three significantly enriched gene modules were phagocytosis, immune response-related terms, and glutamate receptor signalling pathway. We identified novel genes EGF, LPAR5, AGT, AGTR1, RAC2, CD4, CD3D, IL7R, NPY, GRM3, and GRAP2 as potential biomarkers. This study provides valuable information to help understand novel immune evading mechanisms of highly pathogenic NDV-GVII from a host-pathogen interaction point of view. Interactions between the virus and the host determine the success of the viral infection. Hence, the third objective was to identify any particular genes of the virus that may have an important role in virulence and pathogenicity by profiling the transcription of virus in infected birds. This study revealed the transcriptional gradient of these highly pathogenic NDV-GVII genes: NP:P:M:F:HN:L, in which there were a slight attenuations at the NP:P and HN:L gene boundaries. Our result also provides a fully comprehensive qRT-PCR protocol for measuring viral transcript abundance that may be more convenient for laboratories where accessing HTS is not feasible.Thesis (Ph.D.) -- University of Adelaide, School of Animal and Veterinary Sciences, 202
Detection and phasing of single base de novo mutations in biopsies from human in vitro fertilized embryos by advanced whole-genome sequencing
No full textCurrently, the methods available for preimplantation genetic diagnosis (PGD) of in vitro fertilized (IVF) embryos do not detect de novo single-nucleotide and short indel mutations, which have been shown to cause a large fraction of genetic diseases. Detection of all these types of mutations requires whole-genome sequencing (WGS). In this study, advanced massively parallel WGS was performed on three 5- to 10-cell biopsies from two blastocyst-stage embryos. Both parents and paternal grandparents were also analyzed to allow for accurate measurements of false-positive and false-negative error rates. Overall, >95% of each genome was called. In the embryos, experimentally derived haplotypes and barcoded read data were used to detect and phase up to 82% of de novo single base mutations with a false-positive rate of about one error per Gb, resulting in fewer than 10 such errors per embryo. This represents a ∼ 100-fold lower error rate than previously published from 10 cells, and it is the first demonstration that advanced WGS can be used to accurately identify these de novo mutations in spite of the thousands of false-positive errors introduced by the extensive DNA amplification required for deep sequencing. Using haplotype information, we also demonstrate how small de novo deletions could be detected. These results suggest that phased WGS using barcoded DNA could be used in the future as part of the PGD process to maximize comprehensiveness in detecting disease-causing mutations and to reduce the incidence of genetic diseases.Brock A. Peters, Bahram G. Kermani, Oleg Alferov, Misha R. Agarwal, Mark A. McElwain, Natali Gulbahce, Daniel M. Hayden, Y. Tom Tang, Rebecca Yu Zhang, Rick Tearle, Birgit Crain, Renata Prates, Alan Berkeley, Santiago Munné and Radoje Drmana
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