Biology subjects

Buck, D.

Publications and source records attributed to Buck, D..

9 recordsLinked to original sources

bioRxiv2022

Viral burdens are associated with age and viral variant in a population-representative study of SARS-CoV-2 that accounts for time-since-infection related sampling bias.

In this study, we evaluated the impact of viral variant, in addition to other variables, on within-host viral burdens, by analysing cycle threshold (Ct) values derived from nose and throat swabs, collected as part of the UK COVID-19 Infection Survey. Because viral burden distributions determined from community survey data can be biased due to the impact of variant epidemiology on the time-since-infection of samples, we developed a method to explicitly adjust observed Ct value distributions to account for the expected bias. Analysing the adjusted Ct values using partial least squares regression, we found that among unvaccinated individuals with no known prior infection, the average Ct value was 0.94 lower among Alpha variant infections, compared those with the predecessor strain, B.1.177. However, among vaccinated individuals, it was 0.34 lower among Delta variant infections, compared to those with the Alpha variant. In addition, the average Ct value decreased by 0.20 for every 10 year age increment of the infected individual. In summary, within-host viral burdens are associated with age, in addition to the interplay of vaccination status and viral variant.

genetics↗

bioRxiv2021

Using de novo assembly to identify structural variation of complex immune system gene regions

Driven by the necessity to survive environmental pathogens, the human immune system has evolved exceptional diversity and plasticity, to which several factors contribute including inheritable structural polymorphism of the underlying genes. Characterizing this variation is challenging due to the complexity of these loci, which contain extensive regions of paralogy, segmental duplication and high copy-number repeats, but recent progress in long-read sequencing and optical mapping techniques suggests this problem may now be tractable. Here we assess this by using long-read sequencing platforms from PacBio and Oxford Nanopore, supplemented with short-read sequencing and Bionano optical mapping, to sequence DNA extracted from CD14+ monocytes and peripheral blood mononuclear cells from a single European individual identified as HV31. We use this data to build a de novo assembly of eight genomic regions encoding four key components of the immune system, namely the human leukocyte antigen, immunoglobulins, T cell receptors, and killer-cell immunoglobulin-like receptors. Validation of our assembly using k-mer based and alignment approaches suggests that it has high accuracy, with estimated base-level error rates below 1 in 10 kb, although we identify a small number of remaining structural errors. We use the assembly to identify heterozygous and homozygous structural variation in comparison to GRCh38. Despite analyzing only a single individual, we find multiple large structural variants affecting core genes at all three immunoglobulin regions and at two of the three T cell receptor regions. Several of these variants are not accurately callable using current algorithms, implying that further methodological improvements are needed. Our results demonstrate that assessing haplotype variation in these regions is possible given sufficiently accurate long-read and associated data; application of these methods to larger samples would provide a broader catalogue of germline structural variation at these loci, an important step toward making these regions accessible to large-scale genetic association studies.

bioinformatics↗

bioRxiv2020

SARS-CoV-2 genomics beyond the consensus sequence: evidence for circulating mixed viral populations

Extensive global sampling and whole genome sequencing of the pandemic virus SARS-CoV-2 have enabled researchers to characterise its spread, and to identify mutations that may increase transmission or enable the virus to escape therapies or vaccines. Two important components of viral spread are how frequently variants arise within individuals, and how likely they are to be transmitted. Here, we characterise the within-host diversity of SARS-CoV-2, and the extent to which genetic diversity is transmitted, by quantifying variant frequencies in 1390 clinical samples from the UK, many from individuals in known epidemiological clusters. We show that SARS-CoV-2 infections are characterised by low levels of within-host diversity across the entire viral genome, with evidence of strong evolutionary constraint in Spike, a key target of vaccines and antibody-based therapies. Although within-host variants can be observed in multiple individuals in the same phylogenetic or epidemiological cluster, highly infectious individuals with high viral load carry only a limited repertoire of viral diversity. Most viral variants are either lost, or occasionally fixed, at the point of transmission, consistent with a narrow transmission bottleneck. These results suggest potential vaccine-escape mutations are likely to be rare in infectious individuals. Nonetheless, we identified Spike variants present in multiple individuals that may affect receptor binding or neutralisation by antibodies. Since the fitness advantage of escape mutations in highly-vaccinated populations is likely to be substantial, resulting in rapid spread if and when they do emerge, these findings underline the need for continued vigilance and monitoring.

genomics↗

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↗

bioRxiv2020

Rbfox contributes to a conservative program of self-antigen splicing in thymic epithelial cells

Thymic epithelial cells (TEC) control the selection of a T-cell repertoire reactive to pathogens but tolerant of self. While this process is known to involve the promiscuous expression of virtually the entire protein-coding gene repertoire the extent to which TEC recapitulate peripheral isoforms, and the mechanisms by which they do so, have remained largely unknown. We performed the first assembly-based transcriptomic census of transcript structures and splicing factor (SF) expression in mouse medullary TEC (mTEC) and 21 peripheral tissues. Mature mTEC expressed 60.1% of all protein-coding transcripts, more than was detected in any of the peripheral tissues. However, for genes with tissue-restricted expression, we found that mTEC produced fewer isoforms than did the relevant peripheral tissues. Analysis of exon inclusion revealed an absence of brain-specific micro-exons in mTEC. We did not find unusual numbers of novel transcripts in TEC and show that Aire, the facilitator of promiscuous gene expression, promotes usage of long transcripts but has only a limited impact on alternative splicing in mTEC. Comprehensive assessment of SF expression in mTEC identified a small set of non-promiscuously expressed SF genes amongst which we confirmed RBFOX to be present with AIRE in mTEC nuclei. Using a conditional loss of function approach, we show that Rbfox2 promotes mTEC development and regulates the alternative splicing of promiscuously expressed genes. These data indicate that TEC recommission a small number of peripheral SFs, including members of the Rbfox family, to generate a broad but selective representation of the peripheral splice isoform repertoire.

immunology↗

bioRxiv2019

Integrating Transposable Elements in the 3D Genome

Chromosome organisation is increasingly recognised as an essential component of genome regulation, cell fate and cell health. Within the realm of transposable elements (TEs) however, the spatial information of how genomes are folded is still only rarely integrated in experimental studies or accounted for in modelling. Here, we propose a new predictive modelling framework for the study of the integration patterns of TEs based on extensions of widely employed polymer models for genome organisation. Whilst polymer physics is recognised as an important tool to understand the mechanisms of genome folding, we now show that it can also offer orthogonal and generic insights into the integration and distribution profiles (or \"topography\") of TEs across organisms. Here, we present polymer physics arguments and molecular dynamics simulations on TEs inserting into heterogeneously flexible polymers and show with a simple model that polymer folding and local flexibility affects TE integration patterns. The preliminary discussion presented herein lay the foundations for a large-scale analysis of TE integration dynamics and topography as a function of the three-dimensional host genome.

biophysics↗

bioRxiv2018

Single cell RNA-seq reveals profound transcriptional similarity between Barretts esophagus and esophageal glands

Barretts esophagus is a precursor of esophageal adenocarcinoma. In this common condition, squamous epithelium in the esophagus is replaced by columnar epithelium in response to acid reflux. Barretts esophagus is highly heterogeneous and its relationships to normal tissues are unclear. We investigated the cellular complexity of Barretts esophagus and the upper gastrointestinal tract using RNA-sequencing of 2895 single cells from multiple biopsies from four patients with Barretts esophagus and two patients without esophageal pathology. We found that uncharacterised cell populations in Barretts esophagus, marked by LEFTY1 and OLFM4, exhibit a profound transcriptional overlap with a subset of esophageal cells, but not with gastric or duodenal cells. Additionally, SPINK4 and ITLN1 mark cells that precede morphologically identifiable goblet cells in colon and Barretts esophagus, potentially aiding the identification of metaplasia. Our findings reveal striking transcriptional relationships between normal tissue populations and cells in a premalignant condition, with implications for clinical practice.

genetics↗

bioRxiv2018

Low frequency and rare coding variation contributes to multiple sclerosis risk

Multiple sclerosis is a common, complex neurological disease, where almost 20% of risk heritability can be attributed to common genetic variants, including >230 identified by genome-wide association studies (Patsopoulos et al., 2017). Multiple strands of evidence suggest that the majority of the remaining heritability is also due to the additive effects of individual variants, rather than epistatic interactions between these variants, or mutations exclusive to individual families. Here, we show in 68,379 cases and controls that as much as 5% of this heritability is explained by low-frequency variation in gene coding sequence. We identify four novel genes driving MS risk independently of common variant signals, which highlight a key role for regulatory T cell homeostasis and regulation, IFN{gamma} biology and NF{kappa}B signaling in MS pathogenesis. As low-frequency variants do not show substantial linkage disequilibrium with other variants, and as coding variants are more interpretable and experimentally tractable than non-coding variation, our discoveries constitute a rich resource for dissecting the pathobiology of MS.

genetics↗

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↗