Biology subjects

Ragoussis, J.

Publications and source records attributed to Ragoussis, J..

15 recordsLinked to original sources

bioRxiv2025

Integrated Metabolic Complex Genetic Interaction 1 Network of Chromosome 4p Loss in Basal Breast Cancer

Basal breast cancer subtype is enriched for triple-negative breast cancer (TNBC) and exhibits a recurrent large chromosomal deletion in chromosome 4p (chr4p). Chr4p loss is associated with poor survival, evolves early in tumorigenesis and confers on cells a proliferative state. Here, we map the integrated metabolic complex genetic interaction network of chr4p in basal breast cancer to identify targetable vulnerabilities. Differential gene expression analysis of patient derived xenografts and cancer cell models revealed that chr4p loss is associated with changes in cellular energetics and reduction/oxidation balance. Analysis of DepMap pooled genome-wide CRISPR-Cas9 screens identified complex genetic interactions specific to chr4p deletion in basal breast cancer cell models. Functional assays revealed that chr4p loss is associated with disrupted mitochondrial respiratory function and reduced glycolytic capacity, suggesting metabolic rewiring. Increased reactive oxygen species and lipid peroxidation compromised antioxidant defense mechanisms. Ultimately, this study sheds light on targeted therapies for basal breast cancer harboring large chromosomal deletions.

genomics↗

bioRxiv2025

Cerebrospinal fluid-driven ependymal motile cilia defects are implicated in multiple sclerosis pathophysiology

BackgroundMultiple sclerosis is a neurodegenerative autoimmune disorder of the central nervous system (CNS) in which autoreactive immune cells migrate through a damaged blood brain barrier, resulting in focal demyelinating lesions of both the white and grey matter. Of increasing interest is the repeated observation that beyond focal lesions, there are also diffuse, surface-in gradients of pathology in MS, wherein damage is most severe directly adjacent to cerebrospinal fluid (CSF)-contacting surfaces, such as the subpial and periventricular areas. This observation suggests that toxic factors within MS CSF may be contributing to the emergence and/or evolution of surface-in gradients. Directly separating the CSF from the periventricular parenchyma are ependymal cells - a glial epithelium that are equipped with tufts of motile cilia which are critical for circulating CSF solutes and regulating local fluid flow. While damage to ependymal cilia has the potential to drastically modify CSF homeostasis and thus contribute to the damage of CSF exposed regions, these motile cellular structures have yet to be investigated in the context of MS. MethodsWe first conducted single cell RNA sequencing of fresh human periventricular brain tissue containing ependymal cells from MS patients and non-MS disease controls. We subsequently collected CSF from MS patients and exposed cultured rodent ependymal cells to this CSF in order to evaluate impact on ependymal ciliary function. To complement our direct evaluation of cilia in the context of MS, we also confirmed whether cilia were altered in a classic animal model of MS, experimental autoimmune encephalomyelitis (EAE), and also designed a novel transgenic animal model to evaluate the cellular and behavioural effect(s) of adult ependymal ciliary disruption. ResultsSingle cell RNA sequencing analysis of human ependymal cells in MS demonstrated largescale dysregulation of ciliary genes and in situ stains of MS brain tissue confirmed a loss of ependymal cilia. Exposure of ependymal cells to MS CSF led to transcriptional modification of ciliary gene and protein expression and reduced ciliary beating frequency. Likewise, analysis of ependymal cells in EAE also demonstrated altered cilia gene and protein expression. Conditional knockout in adult mice, of the critical cilia-associated gene Ccdc39 in ependymal cells led to transient ventricular enlargement, increased periventricular microglial density, and alterations in nesting behaviour. ConclusionThese data suggest that motile cilia in ependymal cells are dysregulated in CNS autoimmunity. More importantly, however, they provide evidence to suggest that ependymal cilia disruption could play an active role in the development of periventricular pathology in MS and can lead to behavioural deficits that may underlie aspects non-motor MS symptomatology.

neuroscience↗

bioRxiv2025

Finding an optimal sequencing strategy to detect short and long genetic variants in a human genome

Advances in DNA sequencing have transformed genomics, enabling comprehensive insights into human genetic variation. While short-read sequencing (SRS) remains dominant due to its high accuracy and affordability, its limitations in complex genomic regions have spurred the adoption of long-read sequencing (LRS) platforms, such as those from Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT). Despite these advances, there is still a lack of systematic, large-scale benchmarking of variant calling performance across diverse platforms, variant types, sequencing depths, and genomic contexts. Here, we present a comprehensive benchmark of sequencing technologies and variant calling algorithms, evaluating their performance in detecting single nucleotide polymorphisms (SNPs), insertions/deletions (indels), and structural variants (SVs). We show that while SRS combined with DeepVariant or DRAGEN offers excellent small variant detection in well-mapped regions, LRS technologies significantly outperform SRS in complex regions and SV detection. PacBio achieves high SNP and smaller SV accuracy even at moderate coverage, while ONT excels in detecting large SVs. The SV callers Dysgu and SVIM emerged as top performers across LRS datasets. Our results highlight that no single platform is optimal for all variant types or regions: SRS remains optimal for high-throughput small variant detection in accessible regions, whereas LRS is critical for capturing SVs and resolving difficult-to-map loci. These findings offer practical guidance for selecting sequencing technologies, coverage and variant calling strategies tailored to specific research or clinical goals, contributing to more accurate and cost-effective genomic analyses. Highlights[bullet] State-of-the-art short variant calling algorithms are highly comparable but focus on precision and sensitivity differently [bullet]Short-read technologies outperform long-read technologies for small SNP and InDels, with the exception of difficult-to-map variants. [bullet]To capture the majority of variants, a minimum coverage of 15x for PacBio, 20x for SRS, or 30x for ONT is required. However, optimal coverage depends on zygosity, variant type, and the region of interest. [bullet]Long-read technologies outperform short-read technologies for all validation sets tested for deletions and insertions in all size categories.

genomics↗

bioRxiv2025

Single-cell sequencing reveals psilocybin induces sustained cell-type specific plasticity in mouse medial prefrontal cortex

Despite enormous interest in psychedelics for psychiatric interventions, potential underlying biological mechanisms remain unclear. Here, we confirm that a single dose of psilocybin increases synaptic transmission in mouse medial prefrontal cortex. Using scRNA-sequencing, we identify cell-type specific mechanisms of sustained neuroplastic effects. We show that, 24h post-psilocybin, expression of plasticity-related genes is increased in excitatory neurons and that transcription in a type of deep layer near projecting neuron, L5/6 NP, is robustly altered. Analyzing receptor expression patterns reveals that this cell-type specificity does not align with 5-HT2A expression but aligns with 5-HT2C expression patterns. Further, multivariate analyses identify psilocybin-induced gene expression patterns in L5/6 NP neurons predict 5-HT2C, but not 5-HT2A, transcript levels. Pharmacologic manipulation with a 5-HT2C antagonist attenuates the post-acute sustained effect of psilocybin on synaptic transmission, highlighting 5-HT2C signaling and L5/6 NP neurons as key mediators of psychedelic drug actions sustained neuroplastic effects in mPFC.

neuroscience↗

bioRxiv2024

Chromosome-level genome assembly of a doubled haploid brook trout (Salvelinus fontinalis)

Brook trout (Salvelinus fontinalis) is a socioeconomically important fish species for fisheries, aquaculture and aquatic conservation. We produced a 2.5 Gb reference assembly by combining Hi-C chromosome conformation capture with high-coverage short- and long-read sequencing of a fully homozygous mitotic gynogenic doubled haploid fish, which facilitates assembly of highly complex salmonid genomes. The assembly has a N50 of 50.98 Mb and 88.9% of the total assembled sequence length is anchored into 42 main chromosomes, of which 63.44% represents repeated contents, including 1,461,010 DNA transposons. 56,058 genes were found with the NCBI annotation pipeline, with 99% of the 3,640 expected conserved orthologs BUSCO genes (actinopterygii_odb10 lineage database). Additionally, we found significant homology within the 42 chromosomes, as expected for this pseudo-tetraploid species, as well as with the sister species lake trout (Salvelinus namaycush) and Atlantic salmon (Salmo salar). This assembly will serve as a reliable genomic resource for brook trout, thus enabling a wider range of reference-based applications to support ongoing research and management decision-making for the species.

genomics↗

bioRxiv2023

WEGS: a cost-effective sequencing method for genetic studies combining high-depth whole exome and low-depth whole genome

0Whole genome sequencing (WGS) at high-depth (30X) allows the accurate discovery of variants in the coding and non-coding DNA regions and helps elucidate the genetic underpinnings of human health and diseases. Yet, due to the prohibitive cost of high-depth WGS, most large-scale genetic association studies use genotyping arrays or high-depth whole exome sequencing (WES). Here we propose a novel, cost-effective method, which we call "Whole Exome Genome Sequencing" (WEGS), that combines low-depth WGS and high-depth WES with up to 8 samples pooled and sequenced simultaneously (multiplexed). We experimentally assess the performance of WEGS with four different depth of coverage and sample multiplexing configurations. We show that the optimal WEGS configurations are 1.7-2.0 times cheaper than standard WES (no-plexing), 1.8-2.1 times cheaper than high-depth WGS, reach similar recall and precision rates in detecting coding variants as WES, and capture more population-specific variants in the rest of the genome that are difficult to recover when using genotype imputation methods. We apply WEGS to 862 patients with peripheral artery disease and show that it directly assesses more known disease-associated variants than a typical genotyping array and thousands of non-imputable variants per disease-associated locus.

genomics↗

bioRxiv2022

Improved Nanopore full-length cDNA sequencing by PCR-suppression

Full-length transcript sequencing remains a main goal of RNA sequencing. However, even the application of long-read sequencing technologies such as Oxford Nanopore Technologies still fail to yield full-length transcript sequencing for a significant portion of sequenced reads. Since these technologies can sequence reads that are far longer than the longest known processed transcripts, the lack of efficiency to obtain full-length transcripts from good quality RNAs stems from library preparation inefficiency rather than the presence of degraded RNA molecules. It has previously been shown that addition of inverted terminal repeats in cDNA during reverse transcription followed by single-primer PCR creates a PCR suppression effect that prevents amplification of short molecules thus enriching the library for longer transcripts. We adapted this method for Nanopore cDNA library preparation and show that not only is PCR efficiency increased but gene body coverage is dramatically improved. The results show that implementation of this simple strategy will result in better quality full-length RNA sequencing data and make full-length transcript sequencing possible for most of sequenced reads. Contribution to the fieldLong-read RNA sequencing aims to sequence expressed transcripts in their entirety. However, this has remained a challenge, mainly due to inherent inefficiencies in cDNA library preparation. Herein, we provide a new Nanopore cDNA library preparation protocol, termed Panhandle, that improves the efficiency of cDNA PCR with yields 2 - 8 times the yields obtained with ordinary PCR. This is key, as this should in turn reflect in the possibility of lowering the number of PCR cycles needed to obtain ample sequencing material, which in turn could reduce PCR biases, PCR artifacts, turnaround time, reagents, and could increase general quality of the library. Further, transcripts generated using the Panhandle method show better gene body coverage and more accurate transcription start site mapping than regular methods. This represents an important step towards full-length cDNA sequencing by Nanopore.

genomics↗

bioRxiv2021

Transcriptional Reprogramming of Skeletal Muscle Stem Cells by the Niche Environment

Adult stem cells are indispensable for tissue regeneration. Tissue-specific stem cells reside in a specialized location called their niche, where they are in constant cross talk with neighboring niche cells and circulatory signals from their environment. Aging has a detrimental effect on the number and the regenerative function of various stem cells. However, whether the loss of stem cell function is a cause or consequence of their aging niche is unclear. Using skeletal muscle stem cells (MuSCs) as a model, we decouple cell-intrinsic from niche-mediated extrinsic effects of aging on their transcriptome. By combining in vivo MuSC heterochronic transplantation models and computational methods, we show that on a genome-wide scale, age-related altered genes fall into two distinct categories regarding their response to the niche environment. Genes that are inelastic in their response to the niche exhibit altered chromatin accessibility and are associated with differentially methylated regions (DMRs) between young and aged cells. On the other hand, genes that are restorable by niche exposure exhibit altered transcriptome but show no change in chromatin accessibility or DMRs. Taken together, our data suggest that the niche environment plays a decisive role in controlling the transcriptional activity of MuSCs, and exposure to a young niche can reverse approximately half of all age-associated changes that are not epigenetically encoded. The muscle niche therefore serves as an important therapeutic venue to mitigate the negative consequence of aging on tissue regeneration.

genetics↗

bioRxiv2020

Chromosomal rearrangements represent modular cassettes for local adaptation across different geographic scales

Across a species range, multiple sources of environmental heterogeneity, at both small and large scales, create complex landscapes of selection, which may challenge adaptation, particularly when gene flow is high. One key to multidimensional adaptation may reside in the heterogeneity of recombination along the genome. Structural variants, like chromosomal inversions, reduce recombination, increasing linkage disequilibrium among loci at a potentially massive scale. In this study, we examined how chromosomal inversions shape genetic variation across a species range, and ask how their contribution to adaptation in the face of gene flow varies across geographic scales. We sampled the seaweed fly Coelopa frigida along a bioclimatic gradient stretching across 10{degrees} of latitude, a salinity gradient and a range of heterogeneous, patchy habitats. We generated a chromosome-level genome assembly to analyse 1,446 low-coverage whole genomes collected along those gradients. We found several large non-recombining genomic regions, including putative inversions. In contrast to the collinear regions, inversions and low recombining regions differentiated populations more strongly, either along an ecogeographic cline or at a fine-grained scale. These genomic regions were associated with environmental factors and adaptive phenotypes, albeit with contrasting patterns. Altogether, our results highlight the importance of recombination in shaping adaptation to environmental heterogeneity at local and large scales.

evolutionary biology↗

bioRxiv2019

Maleness-on-the-Y (MoY) orchestrates male sex determination in major agricultural fruit fly pests

In insects, rapidly evolving primary sex-determining signals are transduced by a conserved regulatory module producing sex-specific proteins that direct sex determination and sexual differentiation1-4. In the agricultural pest Ceratitis capitata (medfly), a Y-linked maleness factor (M) is thought to repress the autoregulatory splicing of transformer (Cctra), which is required in XX individuals to establish and maintain female sex determination5,6. Despite previous attempts of isolating Y-linked genes using the medfly whole genome, the M factor has remained elusive7. Here, we report the identification of a Y-linked gene, Maleness-on the-Y (MoY), and show that it encodes a small novel protein which is both necessary and sufficient for medfly male sex determination. Transient silencing of MoY in XY individuals leads to the development of fertile females while transient expression of MoY in XX individuals results in fertile males. Notably, a cross between these sex reverted individuals gives rise to both fertile males and females indicating that a functional MoY can be maternally transmitted. In contrast to the diversity of M factors found in dipteran species8-11, we discovered MoY orthologues in seven other Tephritid species spanning [~]111 millions of years of evolution (Mya). We confirmed their male determining function in the olive fly (Bactrocera oleae) and the oriental fruit fly (Bactrocera dorsalis). This unexpected conservation of the primary MoY signal in a large number of important agricultural pests12 will facilitate the development of transferable genetic control strategies in these species, for example sterile male releases or sex-ratio-distorting gene drives.

genetics↗

bioRxiv2018

De novo genome assembly of the olive fruit fly (Bactrocera oleae) developed through a combination of linked-reads and long-read technologies

Long-read sequencing has greatly contributed to the generation of high quality assemblies, albeit at a high cost. It is also not always clear how to combine sequencing platforms. We sequenced the genome of the olive fruit fly (Bactrocera oleae), the most important pest in the olive fruits agribusiness industry, using Illumina short-reads, mate-pairs, 10x Genomics linked-reads, Pacific Biosciences (PacBio), and Oxford Nanopore Technologies (ONT). The 10x linked-reads assembly gave the most contiguous assembly with an N50 of 2.16 Mb. Scaffolding the linked-reads assembly using long-reads from ONT gave a more contiguous assembly with scaffold N50 of 4.59 Mb. We also present the most extensive transcriptome datasets of the olive fly derived from different tissues and stages of development. Finally, we used the Chromosome Quotient method to identify Y-chromosome scaffolds and show that the long-reads based assembly generates very highly contiguous Y-chromosome assembly.\n\nJR is a member of the MinION Access Program (MAP) and has received free-of-charge flow cells and sequencing kits from Oxford Nanopore Technologies for other projects. JR has had no other financial support from ONT.\n\nAB has received re-imbursement for travel costs associated with attending Nanopore Community meeting 2018, a meeting organized my Oxford Nanopore Technologies.

genomics↗

bioRxiv2018

Transcriptome landscape of the developing olive fruit fly embryo delineated by Oxford Nanopore long-read RNA-Seq

The olive fruit fly or olive fly (Bactrocera oleae) is the most important pest of cultivated olive trees. Like all insects the olive fly undergoes complete metamorphosis. However, the transcription dynamics that occur during early embryonic development have not been explored, while detailed transcriptomic analysis in the absence of a fully annotated genome is challenging. We collected olive fly embryos at hourly intervals for the first 6 hours of development and performed full-length cDNA-Seq using a purpose designed SMARTer cDNA synthesis protocol followed by sequencing on the MinION (Oxford Nanopore Technologies). We generated 31 million total reads across the timepoints (median yield 4.2 million per timepoint). The reads showed 98 % alignment rate to the olive fly genome and 91 % alignment rate to the NBCI predicted B. oleae gene models. Over 50 % of the expressed genes had at least one read covering its entire length validating our full-length RNA-Seq procedure. Expression of 68 % of the predicted B. oleae genes was detected in the first six hours of development. We generated a de novo transcriptome assembly of the olive fly and identified 3553 novel genes and a total of 79,810 transcripts; a fourfold increase in transcriptome diversity compared to the NCBI predicted transcriptome. On a global scale, the first six hours of embryo development were characterized by dramatic transcriptome changes with the total number of transcripts per embryo dropping to half from the first hour to the second hour of embryo development. Clustering of genes based on temporal co-expression followed by gene-set enrichment analysiss of genes expressed in the first six hours of embryo development showed that genes involved in transcription and translation, macro-molecule biosynthesis, and neurodevelopment were highly enriched. These data provide the first insight into the transcriptome landscape of the developing olive fly embryo. The data also reveal transcript signatures of sex development. Overall, full-length sequencing of the cDNA molecules permitted a detailed characterization of the isoform complexity and the transcriptional dynamics of the first embryonic stages of the B. oleae.

developmental biology↗

bioRxiv2018

Single-cell RNA-seq reveals that glioblastoma recapitulates normal brain development

SummaryCancer stem cells are critical for cancer initiation, development, and resistance to treatments. Our understanding of these processes, and how they relate to glioblastoma heterogeneity, is limited. To overcome these limitations, we performed single-cell RNA-sequencing on 38 296 glioblastoma cells and 22 637 normal human fetal brain cells. Using an unbiased approach, we mapped the lineage hierarchy of the developing human brain and compared the transcriptome of each cancer cell to this roadmap. We discovered a conserved neural trilineage cancer hierarchy with glial progenitor-like cells at the apex. We also found that this progenitor population contains the majority of cancers cycling cells and is the origin of heterogeneity. Finally, we show that this hierarchal map can be used to identify therapeutic targets specific to progenitor cancer stem cells. Our analyses show that normal brain development reconciles glioblastoma development, unravels the origin of glioblastoma heterogeneity, and helps to identify cancer stem cell-specific targets.

cancer biology↗

bioRxiv2018

Single-nucleus RNA sequencing shows convergent evidence from different cell types for altered synaptic plasticity in major depressive disorder.

Major depressive disorder (MDD) is a complex illness that involves the interaction of different brain systems, pathways, and cell types. Past molecular studies of MDD relied on cellular homogenates of post-mortem brain tissue, making it impossible to determine gene expression changes within individual cells. Using single-cell transcriptomics, we examined almost 80,000 nuclei from the dorsolateral prefrontal cortex of individuals with MDD and healthy controls. Our analyses identified 26 distinct cellular clusters, and over 60% of these showed transcriptional differences between groups. Specifically, 96 genes were differentially expressed, the majority of which were downregulated. Convergent evidence from our analyses, including gene expression, differential correlation, and gene ontology implicated dysregulation of synaptic plasticity in the etiopathogenesis of MDD. Our results show that this high-resolution approach can reveal previously undetectable changes in specific cell types in the context of complex phenotypes and heterogeneous tissues.

neuroscience↗

bioRxiv2018

Custom hereditary breast cancer gene panel selectively amplifies target genes for reliable variant calling

Background: Target enrichment coupled with next generation sequencing provide high-throughput approaches for screening several genes of interest. These approaches facilitate screening a panel of genes for mutations associated with inherited breast cancer for research, diagnostic, and genetic counseling applications.\n\nObjective: To evaluate the performance of our custom 13 gene breast cancer panel, based on singleplex PCR, developed by WaferGen BioSystems. The panel was evaluated using patient-derived DNA samples, in terms of target enrichment efficiency, off-target enrichment, uniformity of target capture, effect of GC content of target regions on coverage depth, and concordance with validated variant calls.\n\nResults: At least 90% of target sequence for each gene was captured at 30x or greater. We evaluated uniformity of target capture across samples by calculating the percentage of samples with at least 90% of total target captured at 100x or greater and found 92% (33/36 samples) uniformity for our panel. Off-target enrichment ranges between 7.2% and 22.3%. We found perfect concordance between our custom panel and the Qiagen human breast cancer panel for functionally annotated variant calls in high read depth shared target regions. Altogether, there was agreement between the panels for 779 variants at 41 loci. We also confirmed 10 pathogenic mutations, initially discovered by Sanger sequencing, in the appropriate samples following target enrichment using our custom WaferGen panel.\n\nConclusion: Our custom hereditary breast cancer panel is sensitive to the desired target genes and facilitates deep sequencing for reliable variant calling.

genetics↗