Biology subjects

Wilkinson, E.

Publications and source records attributed to Wilkinson, E..

9 recordsLinked to original sources

bioRxiv2026

Nanopore sequencing identifies a new Tyr::CreERT2 allele circulating in existing mouse stocks

The Tyr::CreERT2 transgenic mouse lines are essential tools for conditional gene manipulation in melanocytes and are widely used in melanoma research. Two independent lines are in common use: the Bosenberg line and the Larue line. Precise knowledge of transgene integration sites is critical for designing complex genetic crosses, yet the integration sites for these lines have only recently been characterised by whole genome sequencing. Here we report that a Tyr::CreERT2 mouse stock routinely used in BrafCA;Ptenflox melanoma models carries a previously undescribed integration on Chromosome 1, distinct from the previously reported Chromosome 2 integration. Using long-read nanopore sequencing, we mapped the integration to an intergenic locus between Alppl2 and Alpi, revealing a 2,430 bp genomic deletion at the insertion site. We developed position-specific junction PCR and qPCR assays to genotype this allele and confirmed that offspring are born at Mendelian ratios. Given that this stock is associated with elevated spontaneous melanoma penetrance, accurate characterisation of this allele has direct implications for the many laboratories employing this widely distributed melanoma model. SIGNIFICANCEThis study identifies a previously undescribed Tyr::CreERT2 transgene integration on Chromosome 1 in JAX strain 013590, a line widely used across the melanoma research community. Accurate characterisation of this allele has direct practical implications for the many laboratories that rely on this model for studies of melanoma initiation and progression.

genetics↗

bioRxiv2025

Identifying genomic surveillance gaps in Africa for the global public health response to West Nile Virus

BackgroundWest Nile Virus (WNV) is a zoonotic flavivirus of significant One Health relevance and is classified as a priority pathogen with a high-risk of causing public health emergencies of global concern. WNV is endemic to Africa; however, the availability of genomic sequences from the continent remains limited. MethodsWe review the extent of polymerase chain reaction testing and genomic sequencing of WNV conducted across Africa. Using phylogeographic methods, we map the spatiotemporal spread of the virus across the continent and globally. FindingsOur study shows that WNV has been detected in 39 African countries (including Comoros, Seychelles, and Mauritius), the Canary Islands, and Reunion Island. Publications including molecular data originate from 24 countries; however, genomic sequences are publicly available for only 16 countries. We identify regions with detected viral circulation but lacking molecular surveillance. Further, we list such regions that overlap with Key Biodiversity Areas (sites harbouring significant bird diversity) as they may host high viral circulation, and high human population density that may be susceptible to spillover. InterpretationWe recognise significant knowledge gaps on the true disease burden, molecular epidemiology, and distribution of WNV in Africa. Addressing these gaps requires an integrated One Health surveillance approach which is challenging to establish. We propose three key surveillance needs as potential starting points to improve our understanding of the virus in Africa to strengthen the global public health response to this disease. FundingRockefeller Foundation, the National Institute of Health USA, Institute of Human Virology Nigeria, Global Health EDCTP3 Joint Undertaking, the Health Emergency Preparedness and Response Umbrella Program, managed by the World Bank Group, the Medical Research Foundation, and the Wellcome Trust.

molecular biology↗

bioRxiv2023

SeqPanther: Sequence manipulation and mutation statistics toolset

Pathogen genomes harbor critical information necessary to support genomic investigations that inform public health interventions such as treatment, control, and eradication. To extract this information, their sequences are analysed to identify structural variations such as single nucleotide polymorphisms (SNPs) and insertions and deletions (indels) that may be associated with phenotypes of interest. Typically, this involves generating a consensus sequence from raw reads, aligning it to a reference and identifying positions where variations occur. Several pipelines exist to map raw reads and assemble whole genomes for downstream analysis. However, there is no easy to use, freely available bioinformatics quality control (QC) tool to explore mappings for both positional codons and nucleotide distributions in mapped short reads of microbial genomes. To address this problem, we have developed a fast and accurate tool to summarise read counts associated with codons, nucleotides, and indels in mapped next-generation sequencing (NGS) short reads. The tool, developed in Python, also provides a visualization of the genome sequencing depth and coverage. Furthermore, the tool can be run in single or batch mode, where several genomes need to be analysed. Our tool produces a text-based report that enables quick review or can be imported into any analytical tool for upstream analysis. Additionally, the tool also provides functionality to modify the consensus sequences by adding, masking, or restoring to wild type mutations specified by the user. AvailabilitySeqPanther is available at https://github.com/codemeleon/seqPanther, along with the necessary documentation for installation and usage.

bioinformatics↗

bioRxiv2022

Selection analysis identifies unusual clustered mutational changes in Omicron lineage BA.1 that likely impact Spike function

Among the 30 non-synonymous nucleotide substitutions in the Omicron S-gene are 13 that have only rarely been seen in other SARS-CoV-2 sequences. These mutations cluster within three functionally important regions of the S-gene at sites that will likely impact (i) interactions between subunits of the Spike trimer and the predisposition of subunits to shift from down to up configurations, (ii) interactions of Spike with ACE2 receptors, and (iii) the priming of Spike for membrane fusion. We show here that, based on both the rarity of these 13 mutations in intrapatient sequencing reads and patterns of selection at the codon sites where the mutations occur in SARS-CoV-2 and related sarbecoviruses, prior to the emergence of Omicron the mutations would have been predicted to decrease the fitness of any genomes within which they occurred. We further propose that the mutations in each of the three clusters therefore cooperatively interact to both mitigate their individual fitness costs, and adaptively alter the function of Spike. Given the evident epidemic growth advantages of Omicron over all previously known SARS-CoV-2 lineages, it is crucial to determine both how such complex and highly adaptive mutation constellations were assembled within the Omicron S-gene, and why, despite unprecedented global genomic surveillance efforts, the early stages of this assembly process went completely undetected.

evolutionary biology↗

bioRxiv2021

Fingerprinting of skin cells by live cell Raman spectroscopy reveals melanoma cell heterogeneity and cell-type specific responses to UVR

Raman spectroscopy is an emerging dermatological technique with the potential to discriminate biochemically between cell types in a label free and non-invasive manner. Here we use live single cell Raman spectroscopy and principal component analysis (PCA) to fingerprint mouse melanoblasts, melanocytes, keratinocytes and melanoma cells. We show the differences in their spectra are attributable to biomarkers in the melanin biosynthesis pathway and that melanoma cells are a heterogeneous population that sit on a trajectory between undifferentiated melanoblasts and differentiated melanocytes. We demonstrate the utility of Raman spectroscopy as a highly sensitive tool to probe the melanin biosynthesis pathway and its immediate response to UV irradiation revealing previously undescribed opposing responses to UVA and UVB irradiation in melanocytes. Finally, we identify melanocyte specific accumulation of {beta}-carotene correlated with a stabilisation of the UVR response in lipids and proteins consistent with a {beta}-carotene mediated photoprotective mechanism. In summary our data show that Raman spectroscopy can be used to determine the differentiation status of cells of the melanocyte lineage and describe the immediate and temporal biochemical changes associated with UV exposure which differ depending on cell type, differentiation status and competence to synthesise melanin.

cell biology↗

bioRxiv2021

The F-box protein AFF1 regulates ARF protein accumulation to regulate auxin response

Auxin critically regulates nearly every aspect of plant growth and development. Auxin-driven transcriptional responses are mediated through the AUXIN RESPONSE FACTOR (ARF) family of transcription factors. Although ARF protein stability is regulated via the 26S proteasome, molecular mechanisms underlying ARF stability and turnover are unknown. Here, we report the identification and functional characterization of an F-box E3 ubiquitin ligase, which we have named AUXIN RESPONSE FACTOR F-BOX1 (AFF1). AFF1 directly interacts with ARF19 and regulates its accumulation. Mutants defective in AFF1 display ARF19 protein hyperaccumulation, attenuated auxin responsiveness, and developmental defects. Together, our data suggest a new mechanism, namely control of ARF protein stability, in regulating auxin response.

plant biology↗

bioRxiv2020

Whole Genome Sequencing of SARS-CoV-2: Adapting Illumina Protocols for Quick and Accurate Outbreak Investigation During a Pandemic

The COVID-19 pandemic spread very fast around the world. A few days after the first detected case in South Africa, an infection started a large hospital outbreak in Durban, KwaZulu-Natal. Phylogenetic analysis of SARS-CoV-2 genomes can be used to trace the path of transmission within a hospital. It can also identify the source of the outbreak and provide lessons to improve infection prevention and control strategies. In this manuscript, we outline the obstacles we encountered in order to genotype SARS-CoV-2 in real-time during an urgent outbreak investigation. In this process, we encountered problems with the length of the original genotyping protocol, reagent stockout and sample degradation and storage. However, we managed to set up three different library preparation methods for sequencing in Illumina. We also managed to decrease the hands on library preparation time from twelve to three hours, which allowed us to complete the outbreak investigation in just a few weeks. We also fine-tuned a simple bioinformatics workflow for the assembly of high-quality genomes in real-time. In order to allow other laboratories to learn from our experience, we released all of the library preparation and bioinformatics protocols publicly and distributed them to other laboratories of the South African Network for Genomics Surveillance (SANGS) consortium.

genomics↗

bioRxiv2017

Boosting Felsenstein Phylogenetic Bootstrap

Felsensteins article describing the application of the bootstrap to evolutionary trees, is one of the most cited papers of all time. That statistical method, based on resampling and replications, is used extensively to assess the robustness of phylogenetic inferences. However, increasing numbers of sequences are now available for a wide variety of species, and phylogenies with hundreds or thousands of taxa are becoming routine. In that framework, Felsensteins bootstrap tends to yield very low supports, especially on deep branches. We propose a revised version, in which the presence of inferred branches in replications is measured using a gradual \"transfer\" distance, as opposed to the original version using a binary presence/absence index. The resulting supports are higher, while not inducing falsely supported branches. Our method is applied to large simulation, mammal and HIV datasets, for which it reveals the phylogenetic signal, while Felsensteins bootstrap fails to do so.

evolutionary biology↗

bioRxiv2017

External introductions helped drive and sustain the high incidence of HIV-1 in rural KwaZulu-Natal, South Africa

Despite increasing access to antiretroviral therapy, HIV incidence in rural KwaZulu-Natal communities remains among the highest ever reported in Africa. While many epidemiological factors have been invoked to explain this high incidence, widespread human mobility and movement of viral lineages between geographic locations have implicated high rates of transmission across communities. High rates of crosscommunity transmission call into question how effective increasing local coverage of antiretroviral therapy will be at preventing new infections, especially if many new cases arise from external introductions. To help address this question, we use a new phylodynamic modeling approach to estimate both changes in epidemic dynamics through time and the relative contribution of local transmission versus external introductions to overall incidence from HIV-1 subtype C phylogenies. Our phylodynamic estimates of HIV prevalence and incidence are remarkably consistent with population-based surveillance data. Our analysis also reveals that early epidemic dynamics in this population were largely driven by a wave of external introductions. More recently, we estimate that anywhere between 20-60% of all new infections arise from external introductions from outside the local community. These results highlight the power of using phylodynamic methods to study generalized HIV epidemics and the growing need to consider larger-scale regional transmission dynamics above the level of local communities when designing and testing prevention strategies.

epidemiology↗