Biology subjects

Bowden, R.

Publications and source records attributed to Bowden, R..

31 records · Page 2Linked to original sources

bioRxiv2020

A human single-cell atlas of the Substantia nigra reveals novel cell-specific pathways associated with the genetic risk of Parkinson's disease and neuropsychiatric disorders.

We describe a human single-nuclei transcriptomic atlas for the Substantia nigra (SN), generated by sequencing ~ 17,000 nuclei from matched cortical and SN samples. We show that the common genetic risk for Parkinsons disease (PD) is associated with dopaminergic neuron (DaN)-specific gene expression, including mitochondrial functioning, protein folding and ubiquitination pathways. We identify a distinct cell type association between PD risk and oligodendrocyte-specific gene expression. Unlike Alzheimers disease (AD), we find no association between PD risk and microglia or astrocytes, suggesting that neuroinflammation plays a less causal role in PD than AD. Beyond PD, we find associations between SN DaNs and GABAergic neuron gene expression patterns with multiple neuropsychiatric disorders. Nevertheless, we find that each neuropsychiatric disorder is associated with a distinct set of genes within that neuron type. This atlas guides our aetiological understanding by associating SN cell type expression profiles with specific disease risk.

neuroscience↗

bioRxiv2020

Short and long-read genome sequencing methodologies for somatic variant detection; genomic analysis of a patient with diffuse large B-cell lymphoma

Recent advances in throughput and accuracy mean that the Oxford Nanopore Technologies (ONT) PromethION platform is a now a viable solution for genome sequencing. Much of the validation of bioinformatic tools for this long-read data has focussed on calling germline variants (including structural variants). Somatic variants are outnumbered many-fold by germline variants and their detection is further complicated by the effects of tumour purity/subclonality. Here, we evaluate the extent to which Nanopore sequencing enables genome-wide detection and analysis of somatic variation. We do this through sequencing tumour and germline genomes for a patient with diffuse B-cell lymphoma and comparing results with 150bp short-read sequencing of the same samples. Calling germline single nucleotide variants (SNVs) from the long-read data achieved good specificity and sensitivity. However, results of somatic SNV calling highlight the need for the development of specialized joint calling algorithms. We find the comparative performance of different tools varies significantly between structural variant types, and suggest long reads are especially advantageous for calling large somatic deletions and duplications. Finally, we highlight the utility of long reads for phasing clinically relevant variants, confirming that a somatic 1.6Mb deletion and a p.(Arg249Met) mutation involving TP53 are oriented in trans.

genetics↗

bioRxiv2019

Targeted metagenomic sequencing enhances the identification of pathogens associated with acute infection.

The routine identification of pathogens during infection remains challenging because it relies on multiple modalities such as culture and nucleic acid amplification and tests that tend to be specific for very few of an enormous number of possible infectious agents. Metagenomics promises single-test identification, but shotgun sequencing remains unwieldy and expensive or in many cases insufficiently sensitive to detect the amount of pathogen material in a clinical sample. Here we present the validation and application of Castanet, a method for metagenomic sequencing with enrichment that exploits clinical knowledge to construct a broad panel of relevant organisms for detection at low cost with sensitivity comparable to PCR. Castanet targets both DNA and RNA, works with small sample volumes, and can be implemented in a high-throughput diagnostic setting. We used Castanet to analyse plasma samples from 573 patients from the GAinS sepsis cohort and CSF samples from 243 patients from the ChiMES meningitis cohort that had been evaluated using standard clinical microbiology methods, identifying relevant pathogens in many cases where no pathogen had previously been detected. Castanet is intended for use in defining the distribution of pathogens in samples, diseases and populations, for large-scale clinical studies and for verifying the performance of routine testing regimens. By providing sequence as output, Castanet combines pathogen identification directly with subtyping and phylo-epidemiology.

microbiology↗

bioRxiv2018

Illumina and Nanopore methods for whole genome sequencing of hepatitis B virus (HBV)

Advancing interventions to tackle the huge global burden of hepatitis B virus (HBV) infection depends on improved insights into virus epidemiology, transmission, within-host diversity, drug resistance and pathogenesis, all of which can be facilitated by the large-scale generation of full-length virus genome data. Here we describe advances to a protocol to exploit the circular HBV genome structure, using isothermal rolling-circle amplification to enrich for HBV DNA and to generate concatemeric amplicons containing multiple successive copies of the same genome. We show that this product is suitable for Nanopore sequencing as single reads, as well as for generating short-read Illumina sequences. Nanopore reads can be used to implement a straightforward method for error correction that reduces the per-read error rate, by comparing multiple genome copies combined into a single concatemer and by comparing reads generated from plus and minus strands. Thus we can achieve improved consensus sequencing accuracy of 99.7% and resolve intra-sample sequence variants to form whole-genome haplotypes. The combination of isothermal amplification and Nanopore sequencing offers the longer-term potential to develop point-of-care tests for HBV, which could also be adapted for other viruses.

genomics↗

bioRxiv2018

Panton-Valentine leukocidin is the key determinant of Staphylococcus aureus pyomyositis in a bacterial genome-wide association study

Pyomyositis is a severe bacterial infection of skeletal muscle, commonly affecting children in tropical regions and predominantly caused by Staphylococcus aureus. To understand the contribution of bacterial genomic factors to pyomyositis, we conducted a genome-wide association study of S. aureus cultured from 101 children with pyomyositis and 417 children with asymptomatic nasal carriage attending the Angkor Hospital for Children in Cambodia. We found a strong relationship between bacterial genetic variation and pyomyositis, with estimated heritability 63.8% (95% CI 49.2-78.4%). The presence of the Panton-Valentine leucocidin (PVL) locus increased the odds of pyomyositis 130-fold (p =10-17.9). The signal of association mapped both to the PVL-coding sequence and the sequence immediately upstream. Together these regions explained > 99.9% of heritability. Our results establish staphylococcal pyomyositis, like tetanus and diphtheria, as critically dependent on expression of a single toxin and demonstrate the potential for association studies to identify specific bacterial genes promoting severe human disease.

microbiology↗

bioRxiv2018

A comprehensive genomics solution for HIV surveillance and clinical monitoring in a global health setting

High-throughput viral genetic sequencing is needed to monitor the spread of drug resistance, direct optimal antiretroviral regimes, and to identify transmission dynamics in generalised HIV epidemics. Public health efforts to sequence HIV genomes at scale face three major technical challenges: (i) minimising assay cost and protocol complexity, (ii) maximising sensitivity, and (iii) recovering accurate and unbiased sequences of both the genome consensus and the within-host viral diversity. Here we present a novel, high-throughput, virus-enriched sequencing method and computational pipeline tailored specifically to HIV (veSEQ-HIV), which addresses all three technical challenges, and can be used directly on leftover blood drawn for routine CD4 testing. We demonstrate its performance on 1,620 plasma samples collected from consenting individuals attending 10 large urban clinics in Zambia, partners of HPTN 071 (PopART). We show that veSEQ-HIV consistently recovers complete HIV genomes from the majority of samples of different subtypes, and is also quantitative: the number of HIV reads per sample obtained by veSEQ-HIV estimates viral load without the need for additional testing. Both quantitativity and sensitivity were assessed on a subset of 126 samples with clinically measured viral loads, and with standardized quantification controls (VL 100 - 5,000,000 RNA copies/ml). Complete HIV genomes were recovered from 93% (85/91) of samples when viral load was over 1,000 copies per ml. The quantitative nature of the assay implies that variant frequencies estimated with veSEQ-HIV are representative of true variant frequencies in the sample. Detection of minority variants can be exploited for epidemiological analysis of transmission and drug resistance, and we show how the information contained in individual reads of a veSEQ-HIV sample can be used to detect linkage between multiple mutations associated with resistance to antiretroviral therapy. Less than 2% of reads obtained by veSEQ-HIV were identified as in silico contamination events using updates to the phyloscanner software (phyloscanner clean) that we show to be 95% sensitive and 99% specific at decontaminating NGS data. The cost of the assay -- approximately 45 USD per sample -- compares favourably with existing VL and HIV genotyping tests, and provides the additional value of viral load quantification and inference of drug resistance with a single test. veSEQ-HIV is well suited to large public health efforts and is being applied to all [~]9000 samples collected for the HPTN 071-2 (PopART Phylogenetics) study.

genomics↗

bioRxiv2018

Interferon lambda 4 impacts broadly on hepatitis C virus diversity.

Type III interferons (IFN-{lambda}) are part of the innate immune response to hepatitis C virus (HCV) infection however the specific role of IFN-{lambda}4 and the nature of the viral adaption to this pressure have not been defined. Here we use paired genome-wide human and viral genetic data in 485 patients infected with HCV genotype 3a to explore the role of IFN-{lambda}4 on HCV evolution during chronic infection. We show that genetic variations within the host IFNL4 locus have a broad and systematic impact on HCV amino acid diversity. We also demonstrate that this impact is larger in patients producing a more active form of IFN-{lambda}4 protein compared to the less active form. A similar observation was noted for viral load. We conclude that IFN-{lambda}4 protein is a likely causal agent driving widespread HCV amino acid changes and associated with viral load and possibly other clinical and biological outcomes of HCV infection.

genomics↗

bioRxiv2018

Long-read whole genome sequencing and comparative analysis of six strains of the human pathogen Orientia tsutsugamushi

BackgroundOrientia tsutsugamushi is a clinically important but neglected obligate intracellular bacterial pathogen of the Rickettsiaceae family that causes the potentially life-threatening human disease scrub typhus. In contrast to the genome reduction seen in many obligate intracellular bacteria, early genetic studies of Orientia have revealed one of the most repetitive bacterial genomes sequenced to date. The dramatic expansion of mobile elements has hampered efforts to generate complete genome sequences using short read sequencing methodologies, and consequently there have been few studies of the comparative genomics of this neglected species.\n\nResultsWe report new high-quality genomes of Orientia tsutsugamushi, generated using PacBio single molecule long read sequencing, for six strains: Karp, Kato, Gilliam, TA686, UT76 and UT176. In comparative genomics analyses of these strains together with existing reference genomes from Ikeda and Boryong strains, we identify a relatively small core genome of 657 genes, grouped into core gene islands and separated by repeat regions, and use the core genes to infer the first whole-genome phylogeny of Orientia.\n\nConclusionsComplete assemblies of multiple Orientia genomes verify initial suggestions that these are remarkable organisms. They have large genomes with widespread amplification of repeat elements and massive chromosomal rearrangements between strains. At the gene level, Orientia has a relatively small set of universally conserved genes, similar to other obligate intracellular bacteria, and the relative expansion in genome size can be accounted for by gene duplication and repeat amplification. Our study demonstrates the utility of long read sequencing to investigate complex bacterial genomes and characterise genomic variation.

genomics↗

bioRxiv2018

MinION sequencing enables rapid whole genome assembly of Rickettsia typhi in a resource-limited setting

The infrastructure challenges and costs of next-generation sequencing have been largely overcome, for many sequencing applications, by Oxford Nanopore Technologies portable MinION sequencer. However the question remains open whether MinION-based bacterial whole-genome sequencing (WGS) is by itself sufficient for the accurate assessment of phylogenetic and epidemiological relationships between isolates and whether such tasks can be undertaken in resource-limited settings. To investigate this question, we sequenced the genome of an isolate of Rickettsia typhi, an important and neglected cause of fever across much of the tropics and subtropics, for which only three genomic sequences previously existed. We prepared and sequenced libraries on a MinION in Vientiane, Lao PDR using v9.5 chemistry and in parallel we sequenced the same isolate on the Illumina platform in a genomics laboratory in the UK. The MinION sequence reads yielded a single contiguous assembly, in which the addition of Illumina data revealed 226 base-substitution and 5,856 in/del errors. The combined assembly represents the first complete genome sequence of a human R. typhi isolate collected in the last 50 years and differed from the genomes of existing strains collected over a 90-year time period at very few sites, and with no re-arrangements. Filtering based on the known error profile of MinION data improved the accuracy of the Nanopore-only assembly. However, the frequency of false-positive errors remained greater than true sequence divergence from recorded sequences. While Nanopore-only sequencing cannot yet recover phylogenetic signal in R. typhi, such an approach may be applicable for more diverse organisms.

genomics↗

bioRxiv2017

Insights into platypus population structure and history from whole-genome sequencing

The platypus is an egg-laying mammal which, alongside the echidna, occupies a unique place in the mammalian phylogenetic tree. Despite widespread interest in its unusual biology, little is known about its population structure or recent evolutionary history. To provide new insights into the dispersal and demographic history of this iconic species, we sequenced the genomes of 57 platypuses from across the whole species range in eastern mainland Australia and Tasmania. Using a highly-improved reference genome, we called over 6.7M SNPs, providing an informative genetic data set for population analyses. Our results show very strong population structure in the platypus, with our sampling locations corresponding to discrete groupings between which there is no evidence for recent gene flow. Genome-wide data allowed us to establish that 28 of the 57 sampled individuals had at least a third-degree relative amongst other samples from the same river, often taken at different times. Taking advantage of a sampled family quartet, we estimated the de novo mutation rate in the platypus at 7.0x10-9/bp/generation (95% CI 4.1x10-9 - 1.2x10-8/bp/generation). We estimated effective population sizes of ancestral populations and haplotype sharing between current groupings, and found evidence for bottlenecks and long-term population decline in multiple regions, and early divergence between populations in different regions. This study demonstrates the power of whole-genome sequencing for studying natural populations of an evolutionarily important species.

genomics↗

bioRxiv2017

A practical solution for preserving single cells for RNA sequencing

The design and implementation of single-cell experiments is often limited by their requirement for fresh starting material. We have adapted a method for histological tissue fixation using dithio-bis(succinimidyl propionate) (DSP), or Lomants Reagent, to stabilise cell samples for single-cell transcriptomic applications. DSP is a reversible cross-linker of free amine groups that has previously been shown to preserve tissue integrity for histology while maintaining RNA integrity and yield in bulk RNA extractions. Although RNA-seq data from DSP-fixed single cells appears to be prone to characteristic artefacts, such as slightly reduced yield of cDNA and a detectable 3 bias in comparison with fresh cells, cell preservation using DSP does not appear to substantially reduce RNA complexity at the gene level. In addition, there is evidence that instantaneous fixation of cells can reduce inter-cell technical variability. The ability of DSP-fixed cells to retain commonly used dyes, such as propidium iodide, enables the tracking of experimental sub-populations and the recording of cell viability at the point of fixation. Preserving cells using DSP will remove several barriers in the staging of single-cell experiments, including the transport of samples and the scheduling of shared equipment for downstream single-cell isolation and processing.

genomics↗

bioRxiv2017

Severe infections emerge from the microbiome by adaptive evolution

Bacteria responsible for the greatest global mortality colonize the human microbiome far more frequently than they cause severe infections. Whether mutation and selection within the microbiome accompany infection is unknown. We investigated de novo mutation in 1163 Staphylococcus aureus genomes from 105 infected patients with nose-colonization. We report that 72% of infections emerged from the microbiome, with infecting and nose-colonizing bacteria showing parallel adaptive differences. We found 2.8-to-3.6-fold enrichments of protein-altering variants in genes responding to rsp, which regulates surface antigens and toxicity; agr, which regulates quorum-sensing, toxicity and abscess formation; and host-derived antimicrobial peptides. Adaptive mutations in pathogenesis-associated genes were 3.1-fold enriched in infecting but not nose-colonizing bacteria. None of these signatures were observed in healthy carriers nor at the species-level, suggesting disease-associated, short-term, within-host selection pressures. Our results show that infection, like a cancer of the microbiome, emerges through spontaneous adaptive evolution, raising new possibilities for diagnosis and treatment.\n\nOne Sentence SummaryLife-threatening S. aureus infections emerge from nose microbiome bacteria in association with repeatable adaptive evolution.

genomics↗

bioRxiv2017

Resistance genes in global crop breeding networks

Resistance genes are a major tool for managing crop diseases. The crop breeder networks that exchange resistance genes and deploy them in elite varieties help to determine the global landscape of resistance and epidemics, and comprise an important system for maintaining food security. These networks function as complex adaptive systems, with associated strengths and vulnerabilities, and implications for policies to support resistance gene deployment strategies. Extensions of epidemic network analysis can be used to evaluate the multilayer agricultural networks that support and influence crop breeding networks. We evaluate the general structure of crop breeding networks for cassava, potato, rice, and wheat, which illustrate a range of public and private configurations. These systems must adapt to global change in climate and land use, the emergence of new diseases, and disruptive breeding technologies. Principles for maintaining system resilience can be applied to global resistance gene deployment. For example, both diversity and redundancy in the roles played by individual crop breeding groups (public versus private, global versus local) may support societal goals for crop production. Another principle is management of connectivity, where enhanced connectivity among crop breeders may benefit global resistance gene deployment, but increase risks to the durability of resistance genes without effective policies regarding deployment.

genetics↗