Ákos Dörmő

Long-read metagenomic sequencing can improve genome recovery from complex gut microbial communities, yet directly reusable canine gut genome resources remain limited. Here we describe DogMAG, a canine gut metagenome resource based on dog-wise long-read and hybrid assemblies generated by grouping sequencing libraries according to canonical dog identity before assembly. The final dataset comprises 41 assemblies linked to 277 FASTQ records, including 30 Flye long-read-only and 11 OPERA-MS hybrid assemblies. A single integrated BASALT workflow produced 11,276 selected bin/version records, followed by explicit quality-based re-selection of 3,418 medium-quality-or-better metagenome-assembled genome candidates. External dRep dereplication yielded 792 strain-like representatives at 99% average nucleotide identity and 135 species/SGB-like representatives at 95%. GTDB-Tk classified all 792 representatives as Bacteria. Viral screening identified 22,068 geNomad predictions, of which 3,374 Complete, High-quality or Medium-quality viral/proviral candidate rows passed CheckV filtering with contamination ≤10%. DogMAG provides assemblies, genome and viral candidate sequences, metadata, provenance tables and workflow scripts for reuse, benchmarking and reanalysis.

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.

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.

Caviid gammaherpesvirus 1 (CaGHV-1), formerly known as the guinea pig herpes-like virus, is an oncogenic gammaherpesvirus with a sequenced genome but an as-yet uncharacterized transcriptome. Using nanopore long-read RNA sequencing, we annotated the CaGHV-1 genome and constructed a detailed transcriptomic atlas. Our findings reveal diverse viral mRNAs and non-coding RNAs, along with mapped promoter elements for each viral gene. We demonstrated that the CaGHV-1 RTA lytic cycle transcription factor activates its own promoter, similar to Kaposi’s sarcoma-associated herpesvirus (KSHV), and that the CaGHV-1 ORF50 promoter responds to RTA proteins from other gammaherpesviruses, highlighting the evolutionary conservation of RTA-mediated transcriptional mechanisms. Additionally, our analysis uncovered extensive transcriptional overlap within the viral genome, suggesting a role in regulating global gene expression. Given its tumorigenic properties, broad host range, and non-human pathogenicity, this work establishes CaGHV-1 as a promising small animal model for investigating human gammaherpesvirus pathogenesis. IMPORTANCE: The molecular underpinnings of gammaherpesvirus pathogenesis remain poorly understood, partly due to limited animal models. This study provides the first comprehensive transcriptomic atlas of CaGHV-1, highlighting both coding and non-coding RNAs and revealing regulatory elements that drive viral gene expression. Functional studies of the CaGHV-1 RTA transcription factor demonstrated its ability to self-activate and cross-activate promoters from homologous gammaherpesviruses, reflecting conserved mechanisms of transcriptional control. These findings solidify CaGHV-1 as a unique and versatile small animal model, offering new opportunities to investigate gammaherpesvirus replication, transcriptional regulation, and tumorigenesis in a controlled experimental system.

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.

A member of the Varicellovirus genus of herpesviruses, equid alphaherpesvirus 1 (EHV-1) was subjected to timescale profiling in this research. We employed cap analysis gene expression sequencing (CAGE-Seq) on Illumina platform to determine the transcript start sites alongside long-read direct cDNA sequencing (dcDNA-Seq) on Oxford Nanopore Technology platform to detect full-length viral transcripts. Samples were collected at nine distinct stages of the viral lifecycle, with triplicates taken at each stage. We also applied protein synthesis inhibition to determine the immediate-early gene expression of the virus. Analysis of the time-course expression of the viral transcripts using dcDNA-Seq allowed their kinetic categorization. Moreover, the EHV-1 transcriptome was reannotated using CAGE-Seq, long-read dcDNA-Seq, and our previous data obtained from native RNA sequencing.

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.

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.