Biology subjects

Fitzgerald, T.

Publications and source records attributed to Fitzgerald, T..

9 recordsLinked to original sources

bioRxiv2025

Nanopore duplex sequencing reveals patterns of asymmetric states of 5hmC and 5mC in the medaka brain genome

The nucleotide modification 5-methylcytosine (5mC), has been extensively described in terms of its genomic distribution and function. However, the distribution and functional contributions of its oxidised state, 5-hydroxymethylcytosine (5hmC), and its interplay with 5mC remain to be further characterised. Moreover, the hemi-methylation and hemi-hydroxymethylation of CpG dinucleotides in the genome also remain largely unexplored. Here, we use Nanopore duplex sequencing, i.e, the sequencing of both strands of DNA from a single double stranded molecule, to profile the co-occurrence of 5hmC with 5mC in CpG dinucleotides of medaka (japanese rice paddy fish, Oryzias latipes) brain tissues. We characterised asymmetrical patterns of hemi-modified states of CpGs and show using duplex resolution that in the asymmetrical state 5hmC/5mC, 5hmC is preferentially found in the antisense strand of DNA in intron regions, in particular in splice sites, whereas the exons have an enrichment in 5hmC in the sense strand. Furthermore, we found that in the 5hmC/5mC state, 5hmC preferentially occurs along one strand of the DNA duplex in specific families of transposable elements. Altogether these results shed more light on understanding the function and distribution of 5hmC and its mutual role with 5mC in the genome.

genomics↗

bioRxiv2025

Direct RNA nanopore sequencing reveals rapid RNA modification changes following glucose stimulation of human pancreatic beta-cell lines

RNA modifications are critical regulators of gene expression and cellular processes; however, the epitranscriptome is less well studied than the epigenome. Here, we studied transcriptome-wide changes in RNA modifications and expression levels in two human pancreatic beta-cell lines, EndoC-BH1 and EndoC-BH3, after one hour of glucose stimulation. Using direct RNA nanopore sequencing (dRNA-seq), we measured N6-methyladenosine (m6A), 5-methylcytosine (m5C), inosine, and pseudouridine concurrently across the transcriptome. We developed a differential RNA modification method and identified 1,697 differentially modified sites (DMSs) across all modifications. These DMSs were largely independent of changes in gene expression levels and enriched in transcripts for type 2 diabetes (T2D) genes. Our study demonstrates how dRNA-seq can be used to detect and quantify RNA modification changes in response to cellular stimuli at the single-nucleotide level and provides new insights into RNA-mediated mechanisms that may contribute to normal beta-cell response and potential dysfunction in T2D.

genomics↗

bioRxiv2024

Pigmentation and retinal pigment epithelium thickness: a study of the phenotypic and genotypic relationships between ocular and extraocular pigmented tissues.

The retinal pigment epithelium (RPE) is a specialised monolayer of pigmented epithelial cells in the outer retina. The extent to which RPE pigmentation is related to that of other tissues remains unclear. We utilised RPE thickness measured using optical coherence tomography (OCT) imaging as an indicator of RPE melanin content. UK Biobank data was used to assess the relationships between RPE thickness and fundus pigmentation, hair colour, skin colour, and ability to tan. We performed a genome-wide association study (GWAS) to identify genetic loci associated with RPE thickness. We explored the genetic correlation between RPE thickness and pigmentation-related traits. We found that RPE thickness was not phenotypically or globally genetically correlated with hair colour, skin colour or ability to tan. Whilst RPE thickness was phenotypically correlated with fundus pigmentation, there was not significant global genetic correlation. Despite this, variants in key pigmentation loci including TYR, and OCA2-HERC2 were significant in our GWAS of RPE thickness. We identified four genetic regions in which RPE thickness is locally genetically correlated with other pigmentation-related traits, all of which contain protein-coding genes that are central to melanogenesis and melanosome transport. Our study highlights shared and divergent features between RPE thickness and other pigmented traits. SignificanceThe findings outlined in this study support the assertion that pigmentation is related to RPE thickness and show that whilst RPE thicknesss genetic make-up differs from that of other pigmented traits, there is overlap in genes that have critical roles in pigment production. Research HighlightsHere, we explored the relationships between retinal pigment epithelium (RPE) thickness and pigmented traits within and outside of the eye. We found that: (i) well-known pigmentation genes influence RPE thickness; (ii) the thickness of RPE differs according to skin colour; and (iii) RPE thickness is genetically related to other pigmented traits through genes that influence pigmentation.

genetics↗

bioRxiv2024

Measurement and classification of bold-shy behaviours in medaka fish

MotivationBoldness-shyness is considered a fundamental axis of behavioural variation in humans and other species, with obvious adaptive causes and evolutionary implications. Besides an individuals own genetics, this phenotype is also affected by the genetic makeup of peers in the individuals social environment. To identify genetic determinants of variation along the bold-shy behavioural axis, a reliable experimental and analytical setup able to highlight direct and indirect genetic effects is needed. ResultsWe describe a custom assay designed to detect bold-shy behaviours in medaka fish, combining an open-field and novel-object component. We use this assay to explore direct and social genetic effects on the behaviours of 307 pairs of fish from five inbred medaka strains. Applying a Hidden Markov Model (HMM) to classify behavioural modes, we find that direct genetic effects influence the proportions of time the five strains spent in slow-moving states, explaining up to 29.7% of the variance in time spent in those states. We also found that an individuals behaviour is influenced by the genetics of its tank partner, explaining up to 8.64% of the variance in the time spent in slow-moving states. Our behavioural assay in combination with the HMM analysis is applicable to follow-up genetic linkage studies of genetic variants involved in direct behavioural effects and indirect social genetic effects. A suitable genetic resource for such studies, the Medaka Inbred Kiyosu-Karlsruhe panel (MIKK) has recently been established.

bioinformatics↗

bioRxiv2023

Genotype imputation in F2 crosses of inbred lines

MotivationCrosses among inbred lines are a fundamental tool for the discovery of genetic loci associated with phenotypes of interest. In organisms for which large reference panels or SNP chips are not available, imputation from low-pass whole-genome sequencing is an effective method for obtaining genotype data from a large number of individuals. To date, a structured analysis of the conditions required for optimal genotype imputation has not been performed. ResultsWe report a systematic exploration of the effect of several design variables on imputation performance in F2 crosses of inbred medaka lines using the imputation software STITCH. We determined that, depending on the number of samples, imputation performance reaches a plateau when increasing the per-sample sequencing coverage. We also systematically explored the trade-offs between cost, imputation accuracy, and sample numbers. We developed a computational pipeline to streamline the process, enabling other researchers to perform a similar cost-benefit analysis on their population of interest. Availability and implementationThe source code for the pipeline is available at https://github.com/birneylab/stitchimpute. While our pipeline has been developed and tested for an F2 population, the software can also be used to analyse populations with a different structure.

bioinformatics↗

bioRxiv2022

A Poisson reduced-rank regression model for association mapping in sequencing data

Single-cell RNA sequencing (scRNA-seq) technologies allow for the study of gene expression in individual cells. Often, it is of interest to understand how transcriptional activity is associated with cell-specific covariates, such as cell type, genotype, or measures of cell health. Traditional approaches for this type of association mapping assume independence between the outcome variables (or genes), and perform a separate regression for each. However, these methods are computationally costly and ignore the substantial correlation structure of gene expression. Furthermore, count-based scRNA-seq data pose challenges for traditional models based on Gaussian assumptions. We aim to resolve these issues by developing a reduced-rank regression model that identifies low-dimensional linear associations between a large number of cell-specific covariates and high-dimensional gene expression readouts. Our probabilistic model uses a Poisson likelihood in order to account for the unique structure of scRNA-seq counts. We demonstrate the performance of our model using simulations, and we apply our model to a scRNA-seq dataset, a spatial gene expression dataset, and a bulk RNA-seq dataset to show its behavior in three distinct analyses. We show that our statistical modeling approach, which is based on reduced-rank regression, captures associations between gene expression and cell- and sample-specific covariates by leveraging low-dimensional representations of transcriptional states.

bioinformatics↗

bioRxiv2019

RNA modifications detection by comparative Nanopore direct RNA sequencing

RNA molecules undergo a vast array of chemical post-transcriptional modifications (PTMs) that can affect their structure and interaction properties. To date, over 150 naturally occurring PTMs have been identified, however the overwhelming majority of their functions remain elusive. In recent years, a small number of PTMs have been successfully mapped to the transcriptome using experimental approaches relying on high-throughput sequencing. Oxford Nanopore direct-RNA sequencing (DRS) technology has been shown to be sensitive to RNA modifications. We developed and validated Nanocompore, a robust analytical framework to evaluate the presence of modifications in DRS data. To do so, we compare an RNA sample of interest against a non-modified control sample. Our strategy does not require a training set and allows the use of replicates to model biological variability. Here, we demonstrate the ability of Nanocompore to detect RNA modifications at single-molecule resolution in human polyA+ RNAs, as well as in targeted non-coding RNAs. Our results correlate well with orthogonal methods, confirm previous observations on the distribution of N6-methyladenosine sites and provide novel insights into the distribution of RNA modifications in the coding and non-coding transcriptomes. The latest version of Nanocompore can be obtained at https://github.com/tleonardi/nanocompore.

genomics↗

bioRxiv2019

Noncoding microdeletion in mouse Hgf disrupts neural crest migration into the stria vascularis, reduces the endocochlear potential and suggests the neuropathology for human nonsyndromic deafness DFNB39

Hepatocyte growth factor (HGF) is a multifunctional protein that signals through the MET receptor. HGF stimulates cell proliferation, cell dispersion, neuronal survival and wound healing. In the inner ear, levels of HGF must be fine-tuned for normal hearing. In mouse, a deficiency of HGF expression limited to the auditory system, or over-expression of HGF, cause neurosensory deafness. In human, noncoding variants in HGF are associated with nonsyndromic deafness DFNB39. However, the mechanism by which these noncoding variants causes deafness was unknown. Here, we reveal the cause of this deafness using a mouse model engineered with a noncoding intronic 10bp deletion (del10) in Hgf, which is located in the 3UTR of a conserved short isoform (Hgf/NK0.5). Mice homozygous for del10 exhibit moderate-to-profound hearing loss at four weeks of age as measured by pure-tone auditory brainstem responses (ABRs). The wild type +80 millivolt endocochlear potential (EP) was significantly reduced in homozygous del10 mice compared to wild type littermates. In normal cochlea, EPs are dependent on ion homeostasis mediated by the stria vascularis (SV). Previous studies showed that developmental incorporation of neural crest cells into the SV depends on signaling from HGF/MET. We show by immunohistochemistry that in del10 homozygotes, neural crest cells fail to infiltrate the developing SV intermediate layer. Phenotyping and RNAseq analyses reveal no other significant abnormalities in other tissues. We conclude that, in the inner ear, the noncoding del10 mutation in Hgf leads to dysfunctional ion homeostasis in the SV and a loss of EP, recapitulating human DFNB39 deafness.\n\nSignificance StatementHereditary deafness is a common, clinically and genetically heterogeneous neurosensory disorder. Previously we reported that human deafness DFNB39 is associated with noncoding variants in the 3UTR of a short isoform of HGF encoding hepatocyte growth factor. For normal hearing, HGF levels must be fined-tuned as an excess or deficiency of HGF cause deafness in mouse. Using a Hgf mutant mouse with a small 10 base pair deletion recapitulating a human DFNB39 noncoding variant, we demonstrate that neural crest cells fail to migrate into the stria vascularis intermediate layer, resulting in a significantly reduced endocochlear potential, the driving force for sound transduction by inner ear hair cells. HGF-associated deafness is a neurocristopathy but, unlike many other neurocristopathies, it is not syndromic.

genetics↗

bioRxiv2017

ChromoTrace: Reconstruction of 3D Chromosome Configurations by Super-Resolution Microscopy

MotivationThe three-dimensional structure of chromatin plays a key role in genome function, including gene expression, DNA replication, chromosome segregation, and DNA repair. Furthermore the location of genomic loci within the nucleus, especially relative to each other and nuclear structures such as the nuclear envelope and nuclear bodies strongly correlates with aspects of function such as gene expression. Therefore, determining the 3D position of the 6 billion DNA base pairs in each of the 23 chromosomes inside the nucleus of a human cell is a central challenge of biology. Recent advances of super-resolution microscopy in principle enable the mapping of specific molecular features with nanometer precision inside cells. Combined with highly specific, sensitive and multiplexed fluorescence labeling of DNA sequences this opens up the possibility of mapping the 3D path of the genome sequence in situ.\n\nResultsHere we develop computational methodologies to reconstruct the sequence configuration of all human chromosomes in the nucleus from a super-resolution image of a set of fluorescent in situ probes hybridized to the genome in a cell. To test our approach we develop a method for the simulation of chromatin packing in an idealized human nucleus. Our reconstruction method, ChromoTrace, uses suffix trees to assign a known linear ordering of in situ probes on the genome to an unknown set of 3D in situ probe positions in the nucleus from super-resolved images using the known genomic probe spacing as a set of physical distance constraints between probes. We find that ChromoTrace can assign the 3D positions of the majority of loci with high accuracy and reasonable sensitivity to specific genome sequences. By simulating spatial resolution, label multiplexing and noise scenarios we assess algorithm performance under realistic experimental constraints. Our study shows that it is feasible to achieve chromosome-wide reconstruction of the 3D DNA path in chromatin based on super-resolution microscopy images.

bioinformatics↗