Biology subjects

Kakuk, B.

Publications and source records attributed to Kakuk, B..

13 recordsLinked to original sources

bioRxiv2026

Isoform-Resolved Temporal Remodeling of Transcriptome Architecture in Monkeypox Virus

We reanalyzed previously generated replicated Oxford Nanopore direct cDNA sequencing data from six infection time points, together with cap analysis of gene expression and native direct RNA sequencing, to reconstruct full-length monkeypox virus transcripts and characterize their temporal regulation. A broad catalogue of candidate transcript models, including candidate 5' poly(A)-associated structures, was filtered with independent 5'- and 3'-end evidence to define conservative analysis sets. Transcript-resolved profiles revealed a structured temporal program extending beyond gene-level abundance. Host-virus interaction, transcription, and nucleotide-metabolism genes contributed strongly early, whereas virion structure and morphogenesis genes dominated later. Transcription start and end site abundance profiles changed extensively over time, and many genes redistributed among alternative full-length transcript structures. Differential transcript usage was particularly prominent among host-virus interaction genes but depleted among virion structure and morphogenesis genes. Candidate 5' poly(A)-associated transcripts also increased in relative contribution and showed locus-specific patterns. Building on our previous endpoint map, the replicated direct cDNA time course enabled temporal quantification of gene abundance, transcript-end dynamics, and isoform composition. Together, these results show that Monkeypox virus gene regulation involves coordinated remodeling of gene output, transcript boundaries, and full-length transcriptome architecture.

microbiology↗

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

Dog-wise canine gut metagenome assemblies with reconstructed bacterial genomes and viral candidates

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.

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

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

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

Combined Nanopore and Single-Molecule Real-Time Sequencing Survey of Human Betaherpesvirus 5 Transcriptome

Long-read sequencing (LRS), a powerful novel approach, is able to read full-length transcripts and confers a major advantage over the earlier gold standard short-read sequencing in the efficiency of identifying for example polycistronic transcripts and transcript isoforms, including transcript length- and splice variants. In this work, we profile the human cytomegalovirus transcriptome using two third-generation LRS platforms: the Sequel from Pacific BioSciences, and MinION from Oxford Nanopore Technologies. We carried out both cDNA and direct RNA sequencing, and applied the LoRTIA software, developed in our laboratory, for the transcript annotations. This study identified a large number of novel transcript variants, including splice isoforms and transcript start and end site isoforms, as well as putative mRNAs with truncated in-frame ORFs (located within the larger ORFs of the canonical mRNAs), which potentially encode N-terminally truncated polypeptides. Our work also disclosed a highly complex meshwork of transcriptional read-throughs and overlaps.

genomics↗