Biology subjects

Gulyas, G.

Publications and source records attributed to Gulyas, G..

17 recordsLinked to original sources

bioRxiv2026

Taxonomic and functional concordance between full-length ONT 16S and ONT shotgun metagenomics in the canine gut microbiome

Background: Full-length Oxford Nanopore Technologies (ONT) 16S rRNA sequencing provides a scalable view of microbial community composition and can support phylogeny-based functional prediction, but it is not equivalent to shotgun metagenomics. We asked which biological conclusions are preserved when the same canine fecal specimens are profiled by full-length ONT 16S and ONT whole-genome shotgun (WGS) sequencing, and how their agreement depends on analytical scale, reference representation and classifier. Methods: Ninety-seven fecal specimens from 51 dogs were profiled with both assays from the same DNA extract. Functional profiles predicted from NanoASV/NanoPredict with PICRUSt2 were compared with WGS-supported KEGG Ortholog (KO) profiles generated by Kadath. Taxonomy was benchmarked in a source-genome-matched RefSeq universe and in a host-specific DogMAG universe using minitax and Kraken2. Agreement was evaluated at whole-profile, feature-abundance, detection, between-sample structure and biological-inference scales. Age-associated transfer was assessed with dog-aware continuous mixed models, grouped signed-score analyses and paired/dog-blocked PERMANOVA. Results: Functional whole-profile concordance was high: median within-sample CLR Spearman correlations ranged from 0.781 to 0.860 across developmental strata, while between-sample functional structure remained significant by Mantel (rho=0.543) and Procrustes (r=0.693; both p=0.001). Feature-wise transfer was substantially weaker (median KO-wise CLR Spearman=0.318). Continuous age-associated KO slopes showed substantial cross-assay concordance (Spearman=0.727; signed-score Spearman=0.753; direction agreement=77.9%), although 1,290/5,258 eligible KOs retained significant assay-by-age interactions. Taxonomically, exact genus/species abundance agreement was much lower than agreement in between-sample ecological structure. Host-specific DogMAG improved species-level median Spearman from 0.261 to 0.656 for minitax SpeciesEstimate and from 0.181 to 0.512 for Kraken2. The classifier effect was independent of reference choice: under both RefSeq and DogMAG, minitax yielded stronger 16S-WGS concordance than Kraken2, with all eight prespecified RefSeq paired genus/species endpoints and all 10 DogMAG primary paired endpoints significant after BH correction. The same ordering extended to developmental inference, with DogMAG genus/species age-slope concordance of 0.795/0.799 for SpeciesEstimate versus 0.693/0.702 for Kraken2. Taxonomic Aitchison PERMANOVA detected age-associated structure in every assay/reference/classifier/rank combination, whereas age-by-assay interactions were consistently significant but small (R2 approximately 1.1 to 2.2%). Stricter NanoASV identity thresholds removed substantial 16S abundance without improving species-level agreement. Conclusions: The extent of cross-assay agreement depends on the level of analysis. Full-length ONT 16S preserves broad functional organization, ecological structure and much of the direction of age-associated change, but exact fine-rank composition, individual-feature abundance and effect magnitude remain assay dependent. Host-specific reference representation substantially narrows the taxonomic gap, and classifier choice exerts an additional independent effect: within the same matched reference set, minitax consistently yields stronger 16S-WGS concordance than Kraken2 across abundance, detection, ecological-distance and developmental-inference endpoints. Full-length ONT 16S is therefore well suited to broad ecological screening and hypothesis generation, whereas WGS remains preferable when conclusions depend on quantitative fine-rank composition, directly supported gene content or precise feature-level effect estimates.

microbiology↗

bioRxiv2026

Full-length 16S profiling reveals individualized gut microbiota dynamics during short-duration spaceflight

Human spaceflight may perturb the gut microbiota, but densely sampled short missions remain poorly characterized. We profiled 27 phase-matched fecal samples from two astronauts during an 18-day International Space Station mission and one ground-based participant following the same daily schedule using Oxford Nanopore full-length 16S sequencing. Participant identity dominated genus-level Bray-Curtis variation (R2 = 0.489, p < 0.001). In astronaut-only community analyses, mission phase explained 23.4% of genus-level (p = 0.035) and 22.3% of species-level (p = 0.021) variation. Astronauts showed greater displacement from personal baselines than B1 (0.331 versus 0.171) and 1.58-fold higher volatility. Astronaut-only taxon models identified 2 of 81 genera and 5 of 139 species; Collinsella increased from quarantine to orbit (coefficient = 2.586, q = 0.037). Thus, the short-duration spaceflight interval was accompanied by individualized, temporally localized community and taxon shifts rather than uniform microbiota restructuring.

microbiology↗

bioRxiv2026

Early-life canine gut microbiome maturation follows a shared age-diet trajectory within persistent host-specific structure

Early-life gut microbiome maturation remains poorly resolved in dogs. We analyzed 146 fecal samples from 19 Pumi puppies across four dietary stages using paired shotgun metagenomics and Oxford Nanopore full-length 16S sequencing. Bacterial communities shifted from Escherichia-rich facultative assemblages during breastfeeding to anaerobe-rich communities after the transition to solid food, with the strongest compositional change occurring during dietary diversification. Functional profiles changed most strongly at the start of complementary feeding, while viral profiles showed increasing phage richness and stage-dependent predicted phage-host associations. Despite this shared dietary trajectory, host identity explained a substantial fraction of microbiome variation and produced persistent dog-specific abundance patterns. Long-read 16S profiles recovered the main developmental signal despite platform-dependent taxonomic bias, and 16S-based functional inference captured pathway-level contrasts concordant with matched metagenomes. These results identify early-life canine gut microbiome maturation as an age-diet-associated but host-constrained ecological process involving coordinated bacterial, viral, and functional restructuring.

microbiology↗

bioRxiv2025

Longitudinal Long-Read Microbiome Profiling in a Canine Model Reveals How Age, Diet, and Birth Mode Shape Gut Community Dynamics

The gut microbiome undergoes dynamic age-related changes, shaped by diet and maternal factors. Here, we present a species-level, long-read 16S rRNA survey of the developing gut microbiome in a translational canine model, profiling 89 purebred Hungarian Pumis across early-life and reproductive stages. We collected 456 fecal samples longitudinally: 60 puppies followed from birth to 81 weeks, their mothers sampled during pregnancy and lactation, and adult controls from six kennels. We recorded detailed dietary metadata and reproductive status throughout the study. Age was the strongest determinant of alpha diversity, with a rapid increase during weaning and stabilization by six months of age. Beta diversity analyses revealed structured compositional transitions from early developmental phases to adulthood, including a shift toward more uniform, adult-like communities. Within-kennel variation was modest, consistent with shared environmental exposures. Mixed-effects models showed robust associations between specific taxa and age, diet, and kennel, while SparCC-inferred co-occurrence networks indicated increasing ecological complexity with age. We also demonstrated that the delivery mode - vaginal versus cesarean - impacted early-life microbiome composition: Lactobacillus spp. was significantly more abundant in cesarean-born puppies than in vaginally delivered littermates during the 8-10-week window. We also observed reproducible maternal microbiome shifts during pregnancy and lactation, with potential implications for vertical microbial transfer. Taken together, our results show that domestic dogs follow a reproducible, age-structured trajectory of microbial maturation that parallels human development, including delivery-mode effects and diet-responsive taxa. IMPORTANCEMicrobiome research is among the fastest-moving areas in biomedicine, driven by major global efforts to understand how microbial communities shape human health and disease. Dogs provide an ideal translational model because their gut microbiota more closely resembles that of humans than that of other studied animals; moreover, breeds show high within-breed genetic homogeneity, diets can be tightly regulated, and longitudinal sampling across the lifespan is feasible. Mapping shifts driven by diet and maternal factors - from early-life events through later life, including senior stages - is essential to leverage microbial plasticity for prevention, with implications for inflammation, metabolic disease, and neurodegeneration. Here, we advance this goal by providing a longitudinal, high-resolution dataset and demonstrating that full-length 16S rRNA sequencing is a powerful tool for resolving fine-scale patterns of gut colonization and maturation.

microbiology↗

bioRxiv2025

FAST RESENSITIZATION OF G PROTEIN-COUPLED RECEPTORS REQUIRESTHEIR PI(4,5)P2-DEPENDENT SORTING INTO AN AP2 POSITIVE COMPARTMENT

G protein coupled receptors (GPCRs) are the target of about 35% of FDA-approved drugs, which underlines their importance in physiology and disease. Many GPCRs undergo rapid desensitization upon agonist activation, mediated by {beta}-arrestin binding to the phosphorylated receptors, which prevents their G protein coupling. How such receptors regain their G protein signaling competence is poorly understood. Using the AT1 angiotensin II receptor (AT1R), we show that its rapid re-sensitization requires the sorting of the receptors into an AP2-positive plasma membrane (PM) compartment without receptors having to be internalized. This process requires the scaffolding protein, EFR3 and a dedicated PI(4,5)P2 pool specifically produced by PIP5KA. While {beta}-arrestin 1 and -2 both can carry the receptors to the AP2 compartment, weaker binding of {beta}-arrestin 1 to the receptor allows some of the receptors to re-sensitize, while strong {beta}-arrestin 2 binding elicits stronger desensitization and directs the receptors for internalization. These results suggest that the rapid phase of re-sensitization of GPCRs occurs without their endocytosis and primarily takes place at the PM at specific steps during clathrin-coated pit maturation. Together with differences observed between the two {beta}-arrestins to interact with CCPs and the receptors, our data suggest that specific PI(4,5)P2 pools controlled by EFR3A and PIP5K1A determine the balance between {beta}-arrestin1 and -2 receptor interaction and delivery to the AP2 positive compartment, ultimately determining what fraction of the receptors regain their G protein signaling competence.

biochemistry↗

bioRxiv2025

Comprehensive Metagenomic Profiling of Diverse Microbiomes

The canine gut microbiome serves as a key model for veterinary and human health research, but inconsistent findings arise due to methodological variations. This study presents a three-part dataset to clarify how DNA extraction, primer selection, and sequencing platforms influence microbial profiling. First, we performed ultra-deep sequencing of a single dog fecal sample using five DNA isolation kits, multiple library protocols, and four sequencing platforms (Illumina MiSeq/NovaSeq, ONT MinION, PacBio Sequel IIe), enabling direct comparisons of 16S rRNA and shotgun sequencing techniques. Second, we analyzed 40 fecal samples from eight co-housed dogs using Zymo High-Molecular-Weight (ZHMW) and Zymo MagBead (ZMB) extraction kits to assess longitudinal extraction-kit effects. Third, we evaluated three 16S primer systems (standard ONT, PacBio, and modified ONT with degenerate bases) using synthetic mock communities and human/canine fecal samples to quantify primer biases. By integrating synthetic and biological replicates, this dataset provides a standardized resource for benchmarking bioinformatics pipelines and improving cross-study comparability. The study generated 75.31GB of new sequencing data: 43.451GB from ZHMW-ZMB comparisons, 22.611GB for primer evaluations, and 9.191GB from the single-sample analysis. Combined with 31.51GB of prior data, the total dataset exceeds 1061GB, including all analytical outputs. These resources enhance methodological transparency and accuracy in canine gut microbiome research across diverse laboratory workflows.

microbiology↗

bioRxiv2024

Exploring the Transcriptomic Profile of Human Monkeypox Virus via CAGE and Native RNA Sequencing Approaches

In this study, we employed short- and long-read sequencing technologies to delineate the transcriptional architecture of the human monkeypox virus and to identify key regulatory elements that govern its gene expression. Specifically, we conducted a transcriptomic analysis to annotate the transcription start sites (TSSs) and transcription end sites (TESs) of the virus by utilizing cap analysis of gene expression sequencing on the Illumina platform and direct RNA sequencing on the Oxford Nanopore technology device. Our investigations uncovered significant complexity in the use of alternative TSSs and TESs in viral genes. In this research, we also detected the promoter elements and poly(A) signals associated with the viral genes. Additionally, we identified novel genes in both the left and right variable regions of the viral genome. ImportanceGenerally, gaining insight into how the transcription of a virus is regulated offers insights into the key mechanisms that control its life cycle. The recent outbreak of the human monkeypox virus has underscored the necessity of understanding the basic biology of its causative agent. Our results are pivotal for constructing a comprehensive transcriptomic atlas of the human monkeypox virus, providing valuable resources for future studies.

microbiology↗

bioRxiv2023

Cross-Comparison of Gut Metagenomic Profiling Strategies

A critical issue in microbiome research is the selection of reliable laboratory and bioinformatics pipelines. In the absence of generally accepted technical benchmarks and evaluation standards, comparing data generated by different studies becomes challenging. In this work, we carried out the most comprehensive study to date on this topic. We encompassed every stage of processing, from DNA extraction to computational assessment. We adopted four procedures for DNA purification, six for library construction, three for sequencing, and five for bioinformatics. Additionally, we used datasets published by others to corroborate our results. We introduced a software tool that distinctively delivers consistent results, irrespective of sample or dataset origins. This study underscores the importance of methodological optimization at the outset of research projects to ensure the reliability of results and their comparability with findings from other studies. Additionally, this study provides an optimized robust pipeline for gut microbiome analysis.

microbiology↗

bioRxiv2023

Dietary restriction and life-history trade-offs: insights into mTOR pathway regulation and reproductive investment in birds

Resources are needed for growth, reproduction and survival, and organisms must trade-off limited resources among competing processes. Nutritional availability in organisms is sensed and monitored by nutrient-sensing pathways that can trigger physiological changes or alter gene expression. Previous studies have proposed that one such signalling pathway, the mechanistic target of rapamycin (mTOR), underpins a form of adaptive plasticity when individuals encounter constraints in their energy budget. Despite the fundamental importance of this process in evolutionary biology, how nutritional limitation is regulated through the expression of genes governing this pathway and its consequential effects on fitness remains understudied, particularly in birds. We used dietary restriction to simulate resource depletion and examined its effects on body mass, reproduction and gene expression in Japanese quails (Coturnix japonica). Quails were subjected to ad libitum (ADL) feeding or 20%, 30%, and 40% restriction levels for two weeks. All restricted groups exhibited reduced body mass, whereas reductions in the number and mass of eggs were observed only under more severe restrictions. Dietary restriction led to decreased expression of mTOR and insulin-like growth factor 1 (IGF1), whereas the ribosomal protein S6 kinase 1 (RPS6K1) and autophagy-related genes (ATG9A and ATG5) were upregulated. The pattern in which mTOR respond to restriction was similar to what has been seen in body mass. Regardless of the treatment, proportionally higher reproductive investment was associated with individual variation in mTOR expression. These findings reveal the connection between dietary intake and the expression of mTOR and related genes in this pathway.

molecular biology↗

bioRxiv2023

KSHV 3.0: A State-of-the-Art Annotation of the Kaposi's Sarcoma-Associated Herpesvirus Transcriptome Using Cross-Platform Sequencing

Kaposis sarcoma-associated herpesvirus (KSHV) is a large, oncogenic DNA virus belonging to the gammaherpesvirus subfamily. KSHV has been extensively studied with various high-throughput RNA-sequencing approaches to map the transcription start and end sites, the splice junctions, and the translation initiation sites. Despite these efforts, the comprehensive annotation of the viral transcriptome remains incomplete. In the present study, we generated a long-read sequencing dataset of the lytic and latent KSHV transcriptome using native RNA and direct cDNA sequencing methods. This was supplemented with CAGE sequencing based on a short-read platform. We also utilized datasets from previous publications for our analysis. As a result of this combined approach, we have identified a number of novel viral transcripts and RNA isoforms and have either corroborated or improved the annotation of previously identified viral RNA molecules, thereby notably enhancing our comprehension of the transcriptomic architecture of the KSHV genome. We also evaluated the coding capability of transcripts previously thought to be non-coding, by integrating our data on the viral transcripts with translatomic information from other publications. IMPORTANCEDeciphering the viral transcriptome of KSHV is of great importance because we can gain insight into the molecular mechanism of viral replication and pathogenesis, which can help develop potential targets for antiviral interventions. Specifically, the identification of substantial transcriptional overlaps by this work suggests the existence of a genome-wide interference between transcriptional machineries. This finding indicates the presence of a novel regulatory layer, potentially controlling the expression of viral genes.

microbiology↗

bioRxiv2023

Novel Herpesvirus Transcripts with Putative Regulatory Roles in DNA Replication and Global Transcription

In the last couple of years, the rapid advances and decreasing costs of sequencing technologies have revolutionized transcriptomic research. Long-read sequencing (LRS) techniques are able to detect full-length RNA molecules in a single run without the need for additional assembly steps. LRS studies have revealed an unexpected transcriptomic complexity in a variety of organisms, including viruses. A number of transcripts with proven or putative regulatory role, mapping close to or overlapping the replication origins (Oris) and the nearby transcription activator genes, have been described in herpesviruses. In this study, we applied both newly generated and previously published LRS and short-read sequencing datasets to discover additional Ori-proximal transcripts in nine herpesviruses belonging to all of the three subfamilies (alpha, beta and gamma). We identified novel long non-coding RNAs (lncRNAs), as well as splice and length isoforms of mRNAs and lncRNAs. Furthermore, our analysis disclosed an intricate meshwork of transcriptional overlaps at the examined genomic regions. Our results suggest the existence of a super regulatory center, which controls both the replication and the global transcription through multilevel interactions between the molecular machineries.

molecular biology↗

bioRxiv2022

In-depth Temporal Transcriptome Profiling of Monkeypox and Host Cells using Nanopore Sequencing

The recent Monkeypox outbreak showed the importance of studying the basic biology of orthopoxviruses. However, the transcriptome of its causative agent has not been investigated before neither with short-, nor with long-read sequencing approaches. This Oxford Nanopore long-read RNA-Sequencing dataset fills this gap. Our direct cDNA and native RNA sequencing data enable the in-depth characterization of the transcriptomic architecture and dynamics of the gene expressions of monkeypox virus; and also the deeper understanding of the changes it causes in the host cells on a transcriptome level.

microbiology↗

bioRxiv2021

High-Spatiotemporal-Resolution Nanopore Sequencing of SARS-CoV-2 and Host Cell RNAs

Recent studies have disclosed the genome, transcriptome and epigenetic compositions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the effect of viral infection on gene expression of the host cells. It has been demonstrated that, besides the major canonical transcripts, the viral genome also codes for non-canonical RNA molecules. While the structural characterizations have revealed a detailed transcriptomic architecture of the virus, the kinetic studies provided poor and often misleading results on the dynamics of both the viral and host transcripts due to the low temporal resolution of the infection event and the low virus/cell ratio (MOI=0.1) applied for the infection. In this study, we used direct cDNA and direct RNA nanopore sequencings for the generation of high-coverage, high-temporal-resolution transcriptomic datasets on SARS-CoV-2 and on primate host cells infected with a high virus titer (MOI=5). Sixteen sampling time points ranging from 1 to 96h with a varying time resolution and three biological replicates were used in the experiment for both the infected and the non-infected cells.

genomics↗

bioRxiv2021

Palmitoylation targets the Calcineurin phosphatase to the Phosphatidylinositol 4-kinase complex at the plasma membrane

Calcineurin, the conserved protein phosphatase and target of immunosuppressants, is a critical mediator of Ca2+ signaling. To discover novel calcineurin-regulated processes we examined an understudied isoform, CNA{beta}1. We show that unlike canonical cytosolic calcineurin, CNA{beta}1 localizes to the plasma membrane and Golgi due to palmitoylation of its divergent C-terminal tail, which is reversed by the ABHD17A depalmitoylase. Palmitoylation targets CNA{beta}1 to a distinct set of membrane-associated interactors including the phosphatidylinositol 4-kinase (PI4KA) complex containing EFR3B, PI4KA, TTC7B and FAM126A. Hydrogen-deuterium exchange reveals multiple calcineurin-PI4KA complex contacts, including a calcineurin-binding peptide motif in the disordered tail of FAM126A, which we establish as a calcineurin substrate. Calcineurin inhibitors decrease PI4P production during Gq-coupled GPCR signaling, suggesting that calcineurin dephosphorylates and promotes PI4KA complex activity. In sum, this work discovers a new calcineurin-regulated signaling pathway highlighting the PI4KA complex as a regulatory target and revealing that dynamic palmitoylation confers unique localization, substrate specificity and regulation to CNA{beta}1.

biochemistry↗

bioRxiv2020

Time-course Profiling of Bovine Herpesvirus Type 1 and Host Cell Transcriptomes using Multiplatform Sequencing

Long-read sequencing (LRS) has become a standard approach for transcriptome analysis in recent years. This technology is also used for the identification and annotation of genes of various organisms, including viruses. Bovine herpesvirus type 1 (BoHV-1) is an important pathogen of cattle worldwide. However, the transcriptome of this virus is still largely unannotated. This study reports the profiling of the dynamic lytic transcriptome of BoHV-1 using two long-read sequencing (LRS) techniques, the Oxford Nanopore Technology (ONT) MinION, and the Illumina LoopSeq synthetic LRS methods, using multiple library preparation protocols. In this work, we annotated viral mRNAs and non-coding transcripts, and a large number of transcript isoforms, including transcription start and end sites, as well as splice variants of BoHV-1. Very long polycistronic and complex viral transcripts were also detected. Our analysis demonstrated an extremely complex pattern of transcriptional overlaps formed by transcriptional read-throughs or overlapping the 5-untranslated regions of divergently-oriented transcripts. The impact of the viral infection on the host cell transcriptome was also assessed. Our results demonstrate that genes associated with antiviral response as well as viral transcription and translation are upregulated.

genomics↗

bioRxiv2019

Demand for Multiplatform and Meta-analytic Approaches in Transcriptome Profiling

In a recent article, Depledge and colleagues reported a study of the herpes simplex virus type 1 (HSV-1) transcriptome using direct RNA sequencing (dRNA-Seq) on nanopore arrays. The authors provided a useful dataset on full-length viral and host RNA molecules. In this study, we reanalyzed the published dataset and compared it with data generated by our group and others. Our comparative study clearly demonstrated the need for multiplatform and meta-analytic approaches for transcriptome profiling to obtain reliable results.

genomics↗

bioRxiv2019

Multiple Long-read Sequencing Survey of Herpes Simplex Virus Lytic Transcriptome

Long-read sequencing (LRS) has become increasingly important in RNA research due to its strength in resolving complex transcriptomic architectures. In this regard, currently two LRS platforms have demonstrated adequate performance: the Single Molecule Real-Time Sequencing by Pacific Biosciences (PacBio) and the nanopore sequencing by Oxford Nanopore Technologies (ONT). Even though these techniques produce lower coverage and are more error prone than short-read sequencing, they continue to be more successful in identifying transcript isoforms including polycistronic and multi-spliced RNA molecules, as well as transcript overlaps. Recent reports have successfully applied LRS for the investigation of the transcriptome of viruses belonging to various families. These studies have substantially increased the number of previously known viral RNA molecules. In this work, we used the Sequel and MinION technique from PacBio and ONT, respectively, to characterize the lytic transcriptome of the herpes simplex virus type 1 (HSV-1). In most samples, we analyzed the poly(A) fraction of the transcriptome, but we also performed random oligonucleotide-based sequencing. Besides cDNA sequencing, we also carried out native RNA sequencing. Our investigations identified more than 160 previously undetected transcripts, including coding and non-coding RNAs, multi-splice transcripts, as well as polycistronic and complex transcripts. Furthermore, we determined previously unsubstantiated transcriptional start sites, polyadenylation sites, and splice sites. A large number of novel transcriptional overlaps were also detected. Random-primed sequencing revealed that each convergent gene pair produces non-polyadenylated read-through RNAs overlapping the partner genes. Furthermore, we identified novel replication-associated transcripts overlapping the HSV-1 replication origins, and novel LAT variants with very long 5 regions, which are co-terminal with the LAT-0.7kb transcript. Overall, our results demonstrated that the HSV-1 transcripts form an extremely complex pattern of overlaps, and that entire viral genome is transcriptionally active. In most viral genes, if not in all, both DNA strands are expressed.

genomics↗